U.S. patent number 7,348,307 [Application Number 11/416,622] was granted by the patent office on 2008-03-25 for methionine aminopeptidase-2 inhibitors and methods of use thereof.
This patent grant is currently assigned to Praecis Pharmaceuticals Incorporated. Invention is credited to Christopher C. Arico-Muendel, Jens Birktoft, Charles M. Cook, Lily Lee, Barry Morgan, Gary L. Olson, Christopher Self.
United States Patent |
7,348,307 |
Olson , et al. |
March 25, 2008 |
Methionine aminopeptidase-2 inhibitors and methods of use
thereof
Abstract
The present invention provides methods of treating a lymphoma
(e.g., a T-cell lymphoma or a B-cell lymphoma) in a subject by
administering to the subject a therapeutically effective amount of
one or more of the compounds of the invention, for example,
compounds of formulae I and XV: ##STR00001##
Inventors: |
Olson; Gary L. (Mountainside,
NJ), Self; Christopher (West Caldwell, NJ), Lee; Lily
(New York, NY), Cook; Charles M. (Mendham, NJ), Birktoft;
Jens (New York, NY), Morgan; Barry (Franklin, MA),
Arico-Muendel; Christopher C. (West Roxbury, MA) |
Assignee: |
Praecis Pharmaceuticals
Incorporated (Waltham, MA)
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Family
ID: |
46301549 |
Appl.
No.: |
11/416,622 |
Filed: |
May 3, 2006 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20070010452 A1 |
Jan 11, 2007 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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10429174 |
Sep 12, 2006 |
7105482 |
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10138935 |
Jul 19, 2005 |
6919307 |
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10001945 |
Aug 1, 2006 |
7084108 |
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09972772 |
May 2, 2006 |
7037890 |
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09704251 |
Apr 15, 2003 |
6548477 |
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Current U.S.
Class: |
514/19.3;
514/478; 514/588; 514/475; 514/20.1; 514/21.7 |
Current CPC
Class: |
C07D
303/16 (20130101); C07K 5/0817 (20130101); C07K
14/8146 (20130101); C07K 7/06 (20130101); C07K
5/1008 (20130101); C07K 5/1024 (20130101); C07K
5/06078 (20130101); C07K 5/081 (20130101); A61K
38/07 (20130101); A61K 47/64 (20170801); A61K
38/05 (20130101); C07K 5/0806 (20130101); A61K
38/08 (20130101); C07K 5/0202 (20130101); C07K
5/0606 (20130101); A61K 38/4886 (20130101); C07D
405/14 (20130101); Y02A 50/30 (20180101) |
Current International
Class: |
A61K
31/336 (20060101); A61K 38/08 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0354767 |
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Feb 1990 |
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EP |
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0357061 |
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Mar 1990 |
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EP |
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0359036 |
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Mar 1990 |
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EP |
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0387650 |
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Sep 1990 |
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EP |
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0415294 |
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Mar 1991 |
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EP |
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WO-98/13059 |
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Apr 1998 |
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WO |
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WO-98/56372 |
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Dec 1998 |
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WO |
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WO-99/59986 |
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Nov 1999 |
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WO |
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WO-99/61432 |
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Dec 1999 |
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WO |
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WO-00/64486 |
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Nov 2000 |
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WO |
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WO-00/69472 |
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Nov 2000 |
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WO |
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Primary Examiner: Russel; Jeffrey Edwin
Attorney, Agent or Firm: Lahive & Cockfield, LLP
Zacharakis; Maria Laccotripe
Parent Case Text
RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser.
No. 10/429,174, filed May 2, 2003, now U.S. Pat. No. 7,105,482,
issued Sep. 12, 2006; which is a continuation-in-part of U.S.
patent application Ser. No. 10/138,935, filed May 2, 2002, now U.S.
Pat. No. 6,919,307, issued Jul. 19, 2005; which is a
continuation-in-part of U.S. patent application Ser. No.
10/001,945, filed Nov. 1, 2001; now U.S. Pat. No. 7,084,108, issued
Aug. 1, 2006; which is a continuation-in-part of U.S. patent
application Ser. No. 09/972,772, filed Oct. 5, 2001, now U.S. Pat.
No. 7,037,890, issued May 2, 2006; which is a continuation-in-part
of U.S. patent application Ser. No. 09/704,251, filed Nov. 1, 2000,
now U.S. Pat. No. 6,548,477, issued Apr. 15, 2003. The entire
contents of each of the aforementioned applications and patents are
hereby incorporated by reference in their entirety.
Claims
We claim:
1. A method of treating a subject having a lymphoma selected from
the group consisting of precursor (peripheral) T-cell lymphoblastic
lymphoma, adult T-cell lymphoma, extranodal natural killer/T-cell
lymphoma, nasal type lymphoma, enteropathy type T-cell lymphoma,
hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like
T-cell lymphoma, angioimmunoblastic T-cell lymphoma, precursor B
lymphoblastic lymphoma, small lymphocytic lymphoma, B-cell
prolymphocytic lymphoma, lymphoplasmacytic lymphoma, splenic
marginal zone lymphoma, extranodal marginal zone lymphoma, nodal
marginal zone lymphoma, follicular lymphoma, mantle cell lymphoma,
diffuse large B-cell lymphoma, primary mediastinal large B-cell
lymphoma, primary effusion lymphoma, Burkitt's lymphoma, an
AIDS-related lymphoma and a central nervous system lymphoma,
comprising administering to the subject a therapeutically effective
amount of a compound comprising the structure of Formula I,
##STR00062## wherein A is a Met-AP2 inhibitory core; W is O or
NR.sub.2; R.sub.1 and R.sub.2 are each, independently, hydrogen or
alkyl; X is alkylene or substituted alkylene; n is 0 or 1; R.sub.3
and R.sub.4 are each, independently, hydrogen, substituted or
unsubstituted alkyl, substituted or unsubstituted aryl or
substituted or unsubstituted heteroaryl; or R.sub.3 and R.sub.4,
together with the carbon atom to which they are attached, form a
carbocyclic or heterocyclic group; or R.sub.3 and R.sub.4 together
form an alkylene group; Z is --C(O)-- or alkylene-C(O)--; and P is
a peptide comprising from 1 to about 100 amino acid residues
attached at its amino terminus to Z or a group OR.sub.5 or
N(R.sub.6)R.sub.7, wherein R.sub.5, R.sub.6 and R.sub.7 are each,
independently, hydrogen, alkyl, substituted alkyl, azacycloalkyl or
substituted azacycloalkyl; or R.sub.6 and R.sub.7, together with
the nitrogen atom to which they are attached, form a substituted or
unsubstituted heterocyclic ring structure; or Z is --O--,
--NR.sub.8--, alkylene-O-- or alkylene-NR.sub.8--, where R.sub.8 is
hydrogen or alkyl; and P is hydrogen, alkyl or a peptide consisting
of from 1 to about 100 amino acid residues attached at its carboxy
terminus to Z, and pharmaceutically acceptable salts thereof.
2. The method of claim 1, wherein at least one of R.sub.1, R.sub.3
and R.sub.4 is a substituted or unsubstituted alkyl group.
3. The method of claim 2, wherein at least one of R.sub.1, R.sub.3
and R.sub.4 is a substituted or unsubstituted normal, branched or
cyclic C.sub.1-C.sub.6 alkyl group.
4. The method of claim 3, wherein at least one of R.sub.1, R.sub.3
and R.sub.4 is a normal or branched C.sub.1-C.sub.4 alkyl
group.
5. The method of claim 1, wherein one of R.sub.3 and R.sub.4 is a
substituted or unsubstituted aryl group, a substituted or
unsubstituted heteroaryl group, a substituted or unsubstituted
heteroarylalkyl group, or a substituted or unsubstituted aryl alkyl
group.
6. The method of claim 5, wherein one of R.sub.3 and R.sub.4 is
selected from the group consisting of phenyl, naphthyl, indolyl,
imidazolyl, pyridyl, benzyl, naphthylmethyl, indolylmethyl,
imidazolylmethyl and pyridylmethyl.
7. The method of claim 1, wherein n is 1 and X is
C.sub.1-C.sub.6-alkylene.
8. The method of claim 7, wherein X is methylene or ethylene.
9. The method of claim 1, wherein Z is
C.sub.1-C.sub.6-alkylene-C(O)--.
10. The method of claim 9, wherein Z is methylene-C(O)-- or
ethylene-C(O)--.
11. The method of claim 1, wherein at least one of R.sub.6 and
R.sub.7 is alkyl, substituted alkyl, substituted or unsubstituted
azacycloalkyl or substituted or unsubstituted azacycloalkyl.
12. The method of claim 11, wherein at least one of R.sub.6 and
R.sub.7 is an azacycloalkyl group having an N-alkyl
substituent.
13. The method of claim 12, wherein the N-alkyl substituent is a
C.sub.1-C.sub.4-alkyl group.
14. The method of claim 13, wherein the N-alkyl substituent is a
methyl group.
15. The method of claim 1, wherein R.sub.6 and R.sub.7, together
with the nitrogen atom to which they are attached, form a
substituted or unsubstituted five or six-membered aza- or
diazacycloalkyl group.
16. The method of claim 15, wherein R.sub.6 and R.sub.7, together
with the nitrogen atom to which they are attached, form a
substituted or unsubstituted five or six-membered diazacycloalkyl
group which includes an N-alkyl substituent.
17. The method of claim 16, wherein the N-alkyl substituent is a
C.sub.1-C.sub.4-alkyl group.
18. The method of claim 17, wherein the N-alkyl substituent is a
methyl group.
19. The method of claim 1, wherein P is NH.sub.2 or one of the
groups shown below: ##STR00063##
20. A method of treating a subject having a lymphoma selected from
the group consisting of precursor (peripheral) T-cell lymphoblastic
lymphoma, adult T-cell lymphoma, extranodal natural killer/T-cell
lymphoma, nasal type lymphoma, enteropathy type T-cell lymphoma,
hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like
T-cell lymphoma, angioimmunoblastic T-cell lymphoma, precursor B
lymphoblastic lymphoma, small lymphocytic lymphoma, B-cell
prolymphocytic lymphoma, lymphoplasmacytic lymphoma, splenic
marginal zone lymphoma, extranodal marginal zone lymphoma, nodal
marginal zone lymphoma, follicular lymphoma, mantle cell lymphoma,
diffuse large B-cell lymphoma, primary mediastinal large B-cell
lymphoma, primary effusion lymphoma, Burkitt's lymphoma, an
AIDS-related lymphoma and a central nervous system lymphoma,
comprising administering to the subject a therapeutically effective
amount of a compound comprising the structure of Formula XV,
##STR00064## wherein A is a MetAP-2 inhibitory core; W is O or NR;
each R is, independently, hydrogen or alkyl; Z is --C(O)-- or
-alkylene-C(O)--; P is NHR, OR or a peptide consisting of one to
about one hundred amino acid residues connected at the N-terminus
to Z; Q is hydrogen, linear, branched or cyclic alkyl or aryl,
provided that when P is --OR, Q is not hydrogen; or Z is
-alkylene-O-- or -alkylene-N(R)--; P is hydrogen or a peptide
consisting of from one to about one hundred amino acid residues
connected to Z at the carboxyl terminus; Q is hydrogen, linear,
branched or cyclic alkyl or aryl, provided that when P is hydrogen,
Q is not hydrogen; and pharmaceutically acceptable salts
thereof.
21. The method of claim 20, wherein Z is --C(O)-- or
C.sub.1-C.sub.4-alkylene-C(O)--.
22. The method of claim 21, wherein Z is --C(O)-- or
C.sub.1-C.sub.2-alkylene-C(O)--.
23. The method of claim 21, wherein Q is linear, branched or cyclic
C.sub.1-C.sub.6-alkyl, phenyl or naphthyl.
24. The method of claim 23, wherein Q is isopropyl, phenyl or
cyclohexyl.
25. The method of claim 20, wherein Z is
C.sub.1-C.sub.6-alkylene-O-- or C.sub.1-C.sub.6-alkylene-NR--.
26. The method of claim 25, wherein Z is
C.sub.1-C.sub.4-alkylene-O-- or C.sub.1-C.sub.4-alkylene-NH--.
27. The method of claim 26, wherein Z is
C.sub.1-C.sub.2-alkylene-O-- or C.sub.1-C.sub.2-alkylene-NH.
28. The method of claim 25, wherein Q is linear, branched or cyclic
C.sub.1-C.sub.6-alkyl, phenyl or naphthyl.
29. The method of claim 28, wherein Q is isopropyl, phenyl or
cyclohexyl.
30. The method of claim 20, wherein each R is, independently,
hydrogen or linear, branched or cyclic C.sub.1-C.sub.6-alkyl.
31. The method of claim 30, wherein each R is, independently,
hydrogen or linear or branched C.sub.1-C.sub.4-alkyl.
32. The method of claim 31, wherein each R is, independently,
hydrogen or methyl.
33. The method of claim 32, wherein each R is hydrogen.
34. The method of claim 20, wherein A is of Formula II,
##STR00065## wherein R.sub.1' is hydrogen or alkoxy; R.sub.2' is
hydrogen or hydroxy; R.sub.3' is hydrogen or alkyl; and D is linear
or branched alkyl or arylalkyl; or D is of the structure
##STR00066##
35. The method of claim 34, wherein R.sub.1' is
C.sub.1-C.sub.4-alkoxy.
36. The method of claim 35, wherein R.sub.1' is methoxy.
37. The method of claim 34, wherein R.sub.3' is hydrogen or
C.sub.1-C.sub.4-alkyl.
38. The method of claim 37, wherein R.sub.3' is methyl.
39. The method of claim 34, wherein D is linear, branched or cyclic
C.sub.1-C.sub.6-alkyl; or aryl-C.sub.1-C.sub.4-alkyl.
40. The method of claim 20, wherein A is selected from the group
consisting of ##STR00067## wherein p is an integer from 0 to 10;
R.sub.1'' is hydrogen, --OH or C.sub.1-C.sub.4-alkoxy; X'' is a
leaving group; and R.sub.2'' is H, OH, amino,
C.sub.1-C.sub.4-alkylamino or di(C.sub.1-C.sub.4-alkyl)amino).
41. The method of claim 40, wherein A is of the formula
##STR00068##
42. The method of claim 20, wherein P comprises from 1 to about 20
amino acid residues.
43. The method of claim 42, wherein P comprises an amino acid
sequence which is a substrate for a matrix metalloprotease.
44. The method of claim 43, wherein the matrix metalloprotease is
selected from the group consisting of MMP-2, MMP-1, MMP-3, MMP-7,
MMP-8, MMP-9, MMP-12, MMP-13 and MMP-26.
45. The method of claim 44, wherein the matrix metalloprotease is
MMP-2 or MMP-9.
46. The method of claim 45, wherein P comprises the sequence
-Pro-Leu-Gly-Xaa-, wherein Xaa is a naturally occurring amino acid
residue.
47. The method of claim 46, wherein P comprises a sequence selected
from the group consisting of Pro-Cha-Gly-Cys(Me)-His (SEQ ID NO:2);
Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (SEQ ID NO:3);
Pro-Gln-Gly-Ile-Ala-Gly-Trp (SEQ ID NO:4);
Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg (SEQ ID NO:5);
Pro-Leu-Gly-Met-Trp-Ser-Arg (SEQ ID NO:35);
Pro-Leu-Gly-Leu-Trp-Ala-D-Arg (SEQ ID NO:6);
Pro-Leu-Ala-Leu-Trp-Ala-Arg (SEQ ID NO:7);
Pro-Leu-Ala-Leu-Trp-Ala-Arg (SEQ ID NO:8);
Pro-Leu-Ala-Tyr-Trp-Ala-Arg (SEQ ID NO:9);
Pro-Tyr-Ala-Tyr-Trp-Met-Arg (SEQ ID NO:10); Pro-Cha-Gly-Nva-His-Ala
(SEQ ID NO:11); Pro-Leu-Ala-Nva (SEQ ID NO:12); Pro-Leu-Gly-Leu
(SEQ ID NO:13); Pro-Leu-Gly-Ala (SEQ ID NO:14);
Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser (SEQ ID NO:15);
Pro-Cha-Ala-Abu-Cys(Me)-His-Ala (SEQ ID NO:16);
Pro-Cha-Ala-Gly-Cys(Me)-His-Ala (SEQ ID NO:17);
Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu (SEQ ID NO:18);
Pro-Lys-Pro-Leu-Ala-Leu (SEQ ID NO:19);
Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met (SEQ ID NO:20);
Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg (SEQ ID NO:21);
Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg (SEQ ID NO:22); and
Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp (SEQ ID NO:23).
48. A method of treating a subject having a lymphoma selected from
the group consisting of precursor (peripheral) T-cell lymphoblastic
lymphoma, adult T-cell lymphoma, extranodal natural killer/T-cell
lymphoma, nasal type lymphoma, enteropathy type T-cell lymphoma,
hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like
T-cell lymphoma, angioimmunoblastic T-cell lymphoma, precursor B
lymphoblastic lymphoma, small lymphocytic lymphoma, B-cell
prolymphocytic lymphoma, lymphoplasmacytic lymphoma, splenic
marginal zone lymphoma, extranodal marginal zone lymphoma, nodal
marginal zone lymphoma, follicular lymphoma, mantle cell lymphoma,
diffuse large B-cell lymphoma, primary mediastinal large B-cell
lymphoma, primary effusion lymphoma, Burkitt's lymphoma, an
AIDS-related lymphoma and a central nervous system lymphoma,
comprising administering to the subject a therapeutically effective
amount of a compound comprising the structure of formula
##STR00069## wherein W is O or NR'''; each R''' is, independently
hydrogen or a C.sub.1-C.sub.4-alkyl; Q is hydrogen; linear,
branched or cyclic C.sub.1-C.sub.6-alkyl; or aryl; R.sub.1''' is
hydroxy, C.sub.1-C.sub.4-alkoxy or halogen; Z is --C(O)-- or
C.sub.1-C.sub.4-alkylene; P is NHR''', OR''', or a peptide
comprising 1 to 100 amino acid residues attached to Z at the
N-terminus; or Z is alkylene-O or alkylene-NR'''; and P is hydrogen
or peptide comprising 1 to 100 amino acid residues attached to Z at
the C-terminus; or a pharmaceutically acceptable salt thereof;
provided that when P is hydrogen, NHR''' or OR''', Q is not
hydrogen.
49. The method of claim 48, wherein W is O or NH; Z is alkylene-O
or alkylene-NH; Q is isopropyl; R.sub.1''' is methoxy; and P
comprises from 1 to 15 amino acid residues.
50. The method of claim 49, wherein W is O; and P comprises 10 or
fewer amino acid residues.
51. The method of claim 48, wherein P comprises from 1 to about 20
amino acid residues.
52. The method of claim 51, wherein P comprises an amino acid
sequence which is a substrate for a matrix metalloprotease.
53. The method of claim 52, wherein the matrix metalloprotease is
selected from the group consisting of MMP-2, MMP-1, MMP-3, MMP-7,
MMP-8, MMP-9, MMP-12, MMP-13 and MMP-26.
54. The method of claim 53, wherein the matrix metalloprotease is
MMP-2 or MMP-9.
55. The method of claim 54, wherein P comprises the sequence
-Pro-Leu-Gly-Xaa-, wherein Xaa is a naturally occurring amino acid
residue.
56. The method of claim 55, wherein P comprises a sequence selected
from the group consisting of Pro-Cha-Gly-Cys(Me)-His (SEQ ID NO:2);
Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (SEQ ID NO:3);
Pro-Gln-Gly-Ile-Ala-Gly-Trp (SEQ ID NO:4);
Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg (SEQ ID NO:5);
Pro-Leu-Gly-Met-Trp-Ser-Arg (SEQ ID NO:35);
Pro-Leu-Gly-Leu-Trp-Ala-D-Arg (SEQ ID NO:6);
Pro-Leu-Ala-Leu-Trp-Ala-Arg (SEQ ID NO:7);
Pro-Leu-Ala-Leu-Trp-Ala-Arg (SEQ ID NO:8);
Pro-Leu-Ala-Tyr-Trp-Ala-Arg (SEQ ID NO:9);
Pro-Tyr-Ala-Tyr-Trp-Met-Arg (SEQ ID NO:10); Pro-Cha-Gly-Nva-His-Ala
(SEQ ID NO:11); Pro-Leu-Ala-Nva (SEQ ID NO:12); Pro-Leu-Gly-Leu
(SEQ ID NO:13); Pro-Leu-Gly-Ala (SEQ ID NO:14);
Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser (SEQ ID NO:15);
Pro-Cha-Ala-Abu-Cys(Me)-His-Ala (SEQ ID NO:16);
Pro-Cha-Ala-Gly-Cys(Me)-His-Ala (SEQ ID NO:17);
Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu (SEQ ID NO:18);
Pro-Lys-Pro-Leu-Ala-Leu (SEQ ID NO:19);
Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met (SEQ ID NO:20);
Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg (SEQ ID NO:21);
Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg (SEQ ID NO:22); and
Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp (SEQ ID NO:23).
57. A method of treating a subject having a lymphoma selected from
the group consisting of precursor (peripheral) T-cell lymphoblastic
lymphoma, adult T-cell lymphoma, extranodal natural killer/T-cell
lymphoma, nasal type lymphoma, enteropathy type T-cell lymphoma,
hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like
T-cell lymphoma, angioimmunoblastic T-cell lymphoma, precursor B
lymphoblastic lymphoma, small lymphocytic lymphoma, B-cell
prolymphocytic lymphoma, lymphoplasmacytic lymphoma, splenic
marginal zone lymphoma, extranodal marginal zone lymphoma, nodal
marginal zone lymphoma, follicular lymphoma, mantle cell lymphoma,
diffuse large B-cell lymphoma, primary mediastinal large B-cell
lymphoma, primary effusion lymphoma, Burkitt's lymphoma, an
AIDS-related lymphoma and a central nervous system lymphoma,
comprising administering to the subject a therapeutically effective
amount of a compound selected from the group consisting of {(3R,
4S, 5S, 6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylox-
ycarbonylamino}-3-methyl-butyric acid methyl ester; 2-{(3R, 4S, 5S,
6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylox-
ycarbonylamino}-3-methyl-butyric acid methyl ester; 2-{(3R, 4S, 5S,
6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylox-
ycarbonylamino}-4-methyl-pentanoic acid methyl ester; {(3R, 4S, 5S,
6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylox-
ycarbonylamino}-phenyl-acetic acid methyl ester;
(1-Carbamoyl-2-methyl-propyl)-carbamic acid-(3R, 4S, 5S,
6R)-5-methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yl
ester; (1-Carbamoyl-2-methyl-propyl)-carbamic acid-(3R, 4S, 5S,
6R)-5-methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-butyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yl
ester; (1-Hydroxymethyl-2-methyl-propyl)-carbamic acid-(3R, 4S, 5S,
6R)-5-methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yl
ester; 2-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylox-
ycarbonylamino}-3,3-dimethyl-butyric acid methyl ester;
Cyclohexyl-2-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylox-
ycarbonylamino}-acetic acid methyl ester; 2-{(3R, 4S, 5S,
6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylox-
ycarbonylamino}-3-methyl-pentanoic acid methyl ester;
[1-(1-Carbamoyl-2-hydroxy-ethylcarbamoyl)-2-methyl-propyl]-carbamic
acid-(3R, 4S, 5S, 6R)-5-methoxy-4-[(2R,
3R)-2-methyl-3(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yl
ester; 2-(3-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yl}--
ureido)-3-methyl-butyramide; 2-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylox-
ycarbonylamino}-3-methyl-butyric acid;
N-Carbamoyl-Gly-Arg-Gly-Asp-Ser-Pro-(3R, 4S, 5S,
6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-butyl)-oxiranyl]-1-oxa-spir-
o[2.5]oct-6-yl ester (SEQ ID NO:31);
N-Carbamoyl-Gly-Arg-Gly-Asp-Tyr-(OMe)-Arg-Glu-(3R, 4S, 5S,
6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-butyl)-oxiranyl]-1-oxa-spir-
o[2.5]oct-6-yl ester (SEQ ID NO:30);
N-Carbamoyl-Gly-Arg-Gly-Asp-(3R, 4S, 5S,
6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-butyl)-oxiranyl]-1-oxa--
spiro[2.5]oct-6-yl ester (SEQ ID NO:32);
N-Carbamoyl-Gly-Arg-Gly-3-amino-3-pyridyl-propionic acid-(3R, 4S,
5S,
6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-
-spiro[2.5]oct-6-yl ester (SEQ ID NO:40);
N-Carbamoyl-Gly-Pro-Leu-Gly-Met-Trp-Ala-Gly-(3R, 4S, 5S,
6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-
-spiro[2.5]oct-6-yl ester (SEQ ID NO:39);
N-Carbamoyl-Gly-Pro-Leu-(Me)Gly-(3R, 4S, 5S,
6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-
-spiro[2.5]oct-6-yl ester (SEQ ID NO:26);
N-Carbamoyl-Gly-Pro-Leu-Gly-(3R, 4S, 5S,
6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxirany-
l]-1-oxa-spiro[2.5]oct-6-yl ester (SEQ ID NO:27);
Ac-Pro-Leu-Gly-Met-Trp-Ala-(2R-{(3R, 4S, 5S,
6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-
-spiro[2.5]oct-6-yloxycarbonyl}-amino-3-methyl-butanol)ester (SEQ
ID NO:24); Ac-Pro-Leu-Gly-Met-Gly-(2R-{(3R, 4S, 5S
6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-
-spiro[2.5]oct-6-yloxycarbonyl}-amino-3-methyl-butanol)ester (SEQ
ID NO:36); Met-Trp-Ala-(2R-{(3R, 4S, 5S,
6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-
-spiro[2.5]oct-6-yloxycarbonyl}-amino-3-methyl-butanol)ester (SEQ
ID NO:37); Met-Gly-(2R-{(3R, 4S, 5S,
6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-
-spiro[2.5oct-6-yloxycarbonyl]-amino-3-methyl-butanol)ester (SEQ ID
NO:38); Ac-Pro-Leu-Gly-Met-Ala-(2R-{(3R, 4S, 5S,
6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-
-spiro[2.5]oct-6-yloxycarbonyl}-amino-3-methyl-butanol)ester (SEQ
ID NO:34);
{2-Methyl-1-[methyl-(1-methyl-piperidin-4-yl)-carbamoyl]-propyl}--
carbamic acid
5-methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]o-
ct-6-yl ester;
[1-(2-Dimethylamino-ethylcarbamoyl)-2-methyl-propyl]-carbamic acid
5-methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]o-
ct-6-yl ester;
{1-[(2-Dimethylamino-ethyl)-methyl-carbamoyl]-2-methyl-propyl}-carbamic
acid
5-methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[-
2.5]oct-6-yl ester;
[1-(3-Dimethylamino-propylcarbamoyl)-2-methyl-propyl]-carbamic acid
5-methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]o-
ct-6-yl ester;
[1-(3-Dimethylamino-2,2-dimethyl-propylcarbamoyl)-2-methyl-propyl]-carbam-
ic acid
5-methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spir-
o[2.5]oct-6-yl ester;
[2-Methyl-1-(4-methyl-piperazine-1-carbonyl)-propyl]-carbamic acid
5-methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]o-
ct-6-yl ester;
{2-Methyl-1-[2-(1-methyl-pyrrolidin-2-yl)-ethylcarbamoyl]-propyl}-carbami-
c acid
5-methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro-
[2.5]oct-6-yl ester;
[2-Methyl-1-(4-pyrrolidin-1-yl-piperidine-1-carbonyl)-propyl]-carbamic
acid
5-methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[-
2.5]oct-6-yl ester; and
[1-(4-Benzyl-piperazine-1-carbonyl)-2-methyl-propyl]-carbamic acid
5-methoxy-4-[2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]o-
ct-6-yl ester; and pharmaceutically acceptable salts thereof.
58. The method of claim 1, wherein said compound is administered to
the subject using a pharmaceutically acceptable formulation.
59. The method of claim 20, wherein said compound is administered
to the subject using a pharmaceutically acceptable formulation.
60. The method of claim 48, wherein said compound is administered
to the subject using a pharmaceutically acceptable formulation.
61. The method of claim 57, wherein said compound is administered
to the subject using a pharmaceutically acceptable formulation.
62. The method of claim 1, wherein said compound is administered to
the subject intravenously, intramuscularly or orally.
63. The method of claim 20, wherein said compound is administered
to the subject intravenously, intramuscularly or orally.
64. The method of claim 48, wherein said compound is administered
to the subject intravenously, intramuscularly or orally.
65. The method of claim 57, wherein said compound is administered
to the subject intravenously, intramuscularly or orally.
66. The method of claim 1, wherein said subject is human.
67. The method of claim 20, wherein said subject is human.
68. The method of claim 48, wherein said subject is human.
69. The method of claim 57, wherein said subject is human.
70. The method of claim 1, wherein said compound is ##STR00070## or
pharmaceutically acceptable salts thereof.
71. A method of treating a subject having a lymphoma selected from
the group consisting of precursor (peripheral) T-cell lymphoblastic
lymphoma, adult T-cell lymphoma, extranodal natural killer/T-cell
lymphoma, nasal type lymphoma, enteropathy type T-cell lymphoma,
hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like
T-cell lymphoma, angioimmunoblastic T-cell lymphoma, precursor B
lymphoblastic lymphoma, small lymphocytic lymphoma, B-cell
prolymphocytic lymphoma, lymphoplasmacytic lymphoma, splenic
marginal zone lymphoma, extranodal marginal zone lymphoma, nodal
marginal zone lymphoma, follicular lymphoma, mantle cell lymphoma,
diffuse large B-cell lymphoma, primary mediastinal large B-cell
lymphoma, primary effusion lymphoma, Burkitt's lymphoma, an
AIDS-related lymphoma and a central nervous system lymphoma,
comprising administering to said subject an effective amount of a
compound comprising the structure of ##STR00071## or
pharmaceutically acceptable salts thereof.
Description
BACKGROUND OF THE INVENTION
Lymphoma is a leading cause of death in the United States. Lymphoma
is a type of cancer that can occur when an error occurs in the way
a lymphocyte is produced, resulting in an abnormal cell. These
abnormal cells can accumulate by two mechanisms: (a) they can
duplicate faster than normal cells, or (b) they can live longer
than normal lymphocytes. Like normal lymphocytes, the cancerous
lymphocytes can grow in many parts of the body, including the lymph
nodes, spleen, bone marrow, blood, or other organs. There are two
main types of cancer of the lymphatic system. One is called
Hodgkin's disease, while the other is called non-Hodgkin's
lymphoma.
Autoimmune disorders also present a serious health issue in the
United States. A progressive and maintained response by the immune
system against self-components is termed autoimmunity. Normally
self-tolerance mechanisms prevent the immune response from acting
on self-components. However, all mechanisms have a risk of
breakdown and occasionally the immune system turns on its host
environment in an aggressive manner as to cause disease. This
breakdown leads to the copious production of autoreactive B cells
producing autoantibodies and/or autoreactive T cells leading to
destructive autoimmune disease. The cellular mechanisms of
autoimunity are the same as those involved in beneficial immune
responses to foreign components which include antibody-dependent
cell cytotoxicity, delayed-type hypersensitivity (DTH), and T-cell
lympholysis.
Human autoimmune diseases can be divided into two categories:
organ-specific and systemic. In organ-specific autoimmune disease,
autoreactivity is directed to antigens unique to a single organ. In
systemic autoimmune disease, autoreactivity is largely directed
toward a broad range of antigens and involves a number of tissues.
Disease in either type results from the generation of one or both
autoreactive cell types (B or T cells). Autoreactive B cells lead
to the generation of autoantibodies or immune complexes.
Autoreactive T cells lead to the cellular DTH responses from
T.sub.DTh cells or cytotoxic responses from T.sub.C cells.
Diseases caused by parasites are among the leading causes of death
and disease in tropical and subtropical regions of the world.
Efforts to control the invertebrate vector (carrier, such as the
mosquito) of these diseases is, in many cases, difficult as a
result of pesticide resistance, concerns regarding environmental
damage and lack of adequate infrastructure to apply existing vector
control methods. Thus, control of these diseases relies heavily on
the availability of drugs. Unfortunately, most existing
therapeutics are either incompletely effective or toxic to the
human host. In a number of cases, even safe and effective drugs are
failing as a result of the selection and spread of drug resistant
variants of the parasites. This is best dramatized by the global
spread of drug resistant Plasmodium falciparum, the organism
responsible for the most lethal form of malaria.
Angiogenesis is the fundamental process by which new blood vessels
are formed and is essential to a variety of normal body activities
(such as reproduction, development and wound repair). Although the
process is not completely understood, it is believed to involve a
complex interplay of molecules which both stimulate and inhibit the
growth of endothelial cells, the primary cells of the capillary
blood vessels. Under normal conditions, these molecules appear to
maintain the microvasculature in a quiescent state (i.e., one of no
capillary growth) for prolonged periods which may last for as long
as weeks or in some cases, decades. When necessary, however, (such
as during wound repair), these same cells can undergo rapid
proliferation and turnover within a 5 day period (Folkman, J. and
Shing, Y., Journal of Biological Chemistry, 267(16): 10931-10934,
and Folkman, J. and Klagsbrun, M. (1987) Science, 235:
442-447).
Although angiogenesis is a highly regulated process under normal
conditions, many diseases (characterized as "angiogenic diseases")
are driven by persistent unregulated angiogenesis. Otherwise
stated, unregulated angiogenesis may either cause a particular
disease directly or exacerbate an existing pathological condition.
For example, ocular neovacularization has been implicated as the
most common cause of blindness and dominates approximately 20 eye
diseases. In certain existing conditions such as arthritis, newly
formed capillary blood vessels invade the joints and destroy
cartilage. In diabetes, new capillaries formed in the retina invade
the vitreous, bleed, and cause blindness. Growth and metastasis of
solid tumors are also angiogenesis-dependent (Folkman, J. (1986)
Cancer Research 46: 467-473 and Folkman, J. (1989) Journal of the
National Cancer Institute 82: 4-6). It has been shown, for example,
that tumors which enlarge to greater than 2 mm, must obtain their
own blood supply and do so by inducing the growth of new capillary
blood vessels. Once these new blood vessels become embedded in the
tumor, they provide a means for tumor cells to enter the
circulation and metastasize to distant sites, such as the liver,
lung or bone (Weidner, N., et al. (1991) The New England Journal of
Medicine 324(1):1-8).
Fumagillin is a known compound which has been used as an
antimicrobial and antiprotozoal. Its physicochemical properties and
method of production are well known (U.S. Pat. No. 2,803,586 and
Proc. Nat. Acad. Sci. USA (1962) 48:733-735). Fumagillin and
certain types of Fumagillin analogs have also been reported to
exhibit anti-angiogenic activity. However, the use of such
inhibitors (e.g., TNP-470) may be limited by their rapid metabolic
degradation, erratic blood levels, and by dose-limiting central
nervous system (CNS) side effects.
Accordingly, there is still a need for angiogenesis inhibitors
which are more potent, less neurotoxic, more stable, and/or have
longer serum half-lives.
SUMMARY OF THE INVENTION
The present invention provides angiogenesis inhibitor compounds
which comprise a core, e.g., a Fumagillin core, that is believed to
inhibit methionine aminopeptidase 2 (MetAP-2), coupled to a
peptide. The present invention is based, at least in part, on the
discovery that coupling the MetAP-2 inhibitory core to an amino
acid residue or an amino acid derivative prevents the metabolic
degradation of the angiogenesis inhibitor compound to ensure a
superior pharmacokinetic profile and limits CNS side effects by
altering the ability of the angiogenesis inhibitor compound to
cross the blood brain barrier. The present invention is also based,
at least in part, on the discovery that coupling the MetAP-2
inhibitory core to a peptide comprising a site-directed sequence
allows for a cell specific delivery of the angiogenesis inhibitor
compound and limits the toxicity of the angiogenesis inhibitor
compound.
In one aspect the present invention provides a method for treating
a subject (e.g., a mammal, such as a human) suffering from a
lymphoid malignancy. The method includes administering to a subject
an effective amount of a MetAP-2 inhibitor, thereby treating a
subject suffering from a lymphoid malignancy. Lymphoid malignancies
which can be treated with a MetAP-2 inhibitor include lymphoid
leukemias, such as chronic lymphoid leukemia and acute lymphoid
leukemia, and lymphomas, such as T cell lymphoma and B cell
lymphoma.
In a preferred embodiment, the method further includes
administering to the subject a second therapy suitable for treating
a subject suffering from lymphoid malignancy. The second therapy
may be administered to the subject subsequent to, simultaneously or
prior to administration of the MetAP-2 inhibitor to the subject.
The second therapy may include administration of a chemotherapeutic
regimen or a vaccine to the subject.
Accordingly, the present invention provides compounds of Formula
I,
##STR00002##
In Formula I, A is a MetAP-2 inhibitory core, W is O or NR.sub.2,
and R.sub.1 and R.sub.2 are each, independently, hydrogen or alkyl;
X is alkylene or substituted alkylene, preferably linear
C.sub.1-C.sub.6-alkylene; n is 0 or 1; R.sub.3 and R.sub.4 are
each, independently, hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted aryl or arylalkyl or substituted or
unsubstituted heteroaryl or heteroalkyl. R.sub.3 and R.sub.4 can
also, together with the carbon atom to which they are attached,
form a carbocyclic or heterocyclic group; or R.sub.1 and R.sub.4
together can form an alkylene group; Z is --C(O)--, alkylene-C(O)--
or alkylene; and P is a peptide comprising from 1 to about 100
amino acid residues attached at its amino terminus to Z or a group
OR.sub.5 or N(R.sub.6)R.sub.7, wherein R.sub.5, R.sub.6 and R.sub.7
are each, independently, hydrogen, alkyl, substituted alkyl,
azacycloalkyl or substituted azacycloalkyl. R.sub.6 and R.sub.7 can
also form, together with the nitrogen atom to which they are
attached, a substituted or unsubstituted heterocyclic ring
structure.
In another embodiment of the compounds of Formula I, W, X, n,
R.sub.1, R.sub.3 and R.sub.4 have the meanings given above for
these variables; Z is --O--, --NR.sub.8--, alkylene-O-- or
alkylene-NR.sub.8--, where R.sub.8 is hydrogen or alkyl; and P is
hydrogen, alkyl, preferably normal or branched
C.sub.1-C.sub.4-alkyl or a peptide consisting of from 1 to about
100 amino acid residues attached at its carboxy terminus to Z.
In compounds of Formula I, when any of R.sub.1-R.sub.8 is an alkyl
group, preferred alkyl groups are substituted or unsubstituted
normal, branched or cyclic C.sub.1-C.sub.6 alkyl groups.
Particularly preferred alkyl groups are normal or branched
C.sub.1-C.sub.4 alkyl groups. A substituted alkyl group includes at
least one non-hydrogen substituent, such as an amino group, an
alkylamino group or a dialkylamino group; a halogen, such as a
fluoro, chloro, bromo or iodo substituent; or hydroxyl.
When at least one of R.sub.3 and R.sub.4 is a substituted or
unsubstituted aryl or heteroaryl group, preferred groups include
substituted and unsubstituted phenyl, naphthyl, indolyl, imidazoly
and pyridyl. When at least one of R.sub.3 and R.sub.4 is
substituted or unsubstituted arylalkyl or heteroarylalkyl,
preferred groups include substituted and unsubstituted benzyl,
naphthylmethyl, indolylmethyl, imidazolylmethyl and pyridylmethyl
groups. Preferred substituents on aryl, heteroaryl, arylalkyl and
heteroarylalkyl groups are independently selected from the group
consisting of amino, alkyl-substituted amino, halogens, such as
fluoro, chloro, bromo and iodo; hydroxyl groups and alkyl groups,
preferably normal or branched C.sub.1-C.sub.6-alkyl groups, most
preferably methyl groups. X is preferably linear
C.sub.1-C.sub.6-alkylene, more preferably C.sub.1-C.sub.4-alkylene
and most preferably methylene or ethylene. When Z is
alkylene-C(O)--, alkylene-O-- or alkylene-NR.sub.8, the alkylene
group is preferably linear C.sub.1-C.sub.6-alkylene, more
preferably C.sub.1-C.sub.4-alkylene and most preferably methylene
or ethylene.
R.sub.6 and R.sub.7, in addition to alkyl, substituted alkyl or
hydrogen, can each also independently be a substituted or
unsubstituted azacycloalkyl group or a substituted or unsubstituted
azacycloalkylalkyl group. Suitable substituted azacycloalkyl groups
include azacycloalkyl groups which have an N-alkyl substituent,
preferably an N--C.sub.1-C.sub.4-alkyl substituent and more
preferably an N-methyl substituent. R.sub.6 and R.sub.7 can also,
together with the nitrogen atom to which they are attached, form a
heterocyclic ring system, such as a substituted or unsubstituted
five or six-membered aza- or diazacycloalkyl group. Preferably, the
diazacycloalkyl group includes an N-alkyl substituent, such as an
N--C.sub.1-C.sub.4-alkyl substituent or, more preferably, an
N-methyl substituent.
In particularly preferred embodiments, --N(R.sub.6)R.sub.7 is
NH.sub.2 or one of the groups shown below:
##STR00003##
Preferably, the compounds of Formula I do not include compounds
wherein Z is --O--, P is hydrogen, R.sub.3 and R.sub.4 are both
hydrogen, n is 1 and X is methylene. Preferably, the compounds of
Formula I further do not include compounds wherein Z is
methylene-O--, R.sub.3 and R.sub.4 are both hydrogen, and n is
0.
In another aspect, the present invention is directed to
angiogenesis inhibitor compounds of Formula XV,
##STR00004## where A is a MetAP-2 inhibitory core and W is O or NR.
In one embodiment, Z is --C(O)-- or -alkylene-C(O)-- and P is NHR,
OR or a peptide consisting of one to about one hundred amino acid
residues connected at the N-terminus to Z. In this embodiment, Q is
hydrogen, linear, branched or cyclic alkyl or aryl, provided that
when P is --OR, Q is not hydrogen.
In another embodiment, Z is -alkylene-O-- or -alkylene-N(R)-- and P
is hydrogen or a peptide consisting of from one to about one
hundred amino acid residues connected to Z at the carboxyl
terminus. In this embodiment, Q is hydrogen, linear, branched or
cyclic alkyl or aryl, provided that when P is hydrogen, Q is not
hydrogen.
In the angiogenesis inhibitor compounds of Formula XV, each R is,
independently, hydrogen or alkyl.
In another aspect, the invention features pharmaceutical
compositions comprising the angiogenesis inhibitor compounds of
Formula I or XV and a pharmaceutically acceptable carrier.
In yet another aspect, the invention features a method of treating
an angiogenic disease, e.g., cancer (such as lung cancer, brain
cancer, kidney cancer, colon cancer, liver cancer, pancreatic
cancer, stomach cancer, prostate cancer, breast cancer, ovarian
cancer, cervical cancer, melanoma, and metastatic versions of any
of the preceding cancers), in a subject. The method includes
administering to the subject a therapeutically effective amount of
one or more angiogenesis inhibitor compounds of Formula I or
XV.
In one embodiment, the present invention provides a method of
treating a subject suffering from a parasitic infection, such as an
infection by Plasmodium species, such as Plasmodium falciparum, or
an infection by Leishmania species, such as Leishmania donavani.
The method comprises the step of administering to the subject a
therapeutically effective amount of a compound of the invention.
The subject can be an individual who is suffering from, or
susceptible to, infection by a parasitic organism. In a preferred
embodiment, the subject suffers from malaria or Leishmaniasis.
The invention further provides a method of treating a subject
suffering from a lymphoid malignancy. The method comprises the step
of administering to the subject a therapeutically effective amount
of a compound of the invention. Suitable lymphoid malignancies
which can be treated with a compound of the invention include
lymphoid leukemias, such as chronic lymphoid leukemia and acute
lymphoid leukemia, and lymphomas, such as Non-Hodgkin's lymphoma,
including T cell lymphoma and B cell lymphoma.
In a further embodiment, the invention provides a method of
treating a subject suffering from an autoimmune disorder,
comprising the step of administering to the subject a
therapeutically effective amount of a compound of the invention.
The autoimmune disorder can be, for example, rheumatoid arthritis,
lupus erythematosus, psoriasis, multiple sclerosis, myasthenia
gravis, vasculitis, or diabetes mellitus.
Other features and advantages of the invention will be apparent
from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a series of graphs depicting the inhibition of SR cell
proliferation in culture following 3 or 6 days of exposure to
Compound 5 (representative data).
FIG. 2 is a graph depicting tumor volumes of SR lymphoma
tumor-bearing mice treated with Compound 5.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides compounds useful as angiogenesis
inhibitors and methods for using these compounds in the treatment
of angiogenic diseases. Without intending to be limited by theory,
it is believed that the angiogenesis inhibitor compounds of the
invention inhibit angiogenesis by inhibiting methionine
aminopeptidase 2 (MetAP-2), an enzyme which cleaves the N-terminal
methionine residue of newly synthesized proteins to produce the
active form of the protein. At the same time, the presence of a
peptide in the angiogenesis inhibitor compounds of the invention
prevents the metabolic degradation of the angiogenesis inhibitor
compounds and ensures a superior pharmacokinetic profile. The
presence of the peptide in the angiogenesis inhibitor compounds of
the invention also alters the ability of the angiogenesis inhibitor
compound to cross the blood brain barrier to, for example, limit
CNS side effects (such as CNS toxicity). The presence of peptides
comprising a site-directed sequence in the angiogenesis inhibitor
compounds of the invention allows for a site-specific delivery of
the angiogenesis inhibitor compounds and, thus, limits the toxicity
of the angiogenesis inhibitor compounds.
The angiogenesis inhibitor compounds of the invention comprise a
MetAP-2 inhibitory core and a peptide attached, directly or
indirectly, thereto. In one embodiment, the invention provides
angiogenesis inhibitor compounds of Formula I
##STR00005##
In Formula I, A is a MetAP-2 inhibitory core, W is O or NR.sub.2,
and R.sub.1 and R.sub.2 are each, independently, hydrogen or alkyl;
X is alkylene or substituted alkylene, preferably linear
C.sub.1-C.sub.6-alkylene; n is 0 or 1; R.sub.3 and R.sub.4 are
each, independently, hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted aryl or arylalkyl or substituted or
unsubstituted heteroaryl or heteroalkyl. R.sub.3 and R.sub.4 can
also, together with the carbon atom to which they are attached,
form a carbocyclic or heterocyclic group; or R.sub.1 and R.sub.4
together can form an alkylene group; Z is --C(O)--, alkylene-C(O)--
or alkylene; and P is a peptide comprising from 1 to about 100
amino acid residues attached at its amino terminus to Z or a group
OR.sub.5 or N(R.sub.6)R.sub.7, wherein R.sub.5, R.sub.6 and R.sub.7
are each, independently, hydrogen, alkyl, substituted alkyl,
azacycloalkyl or substituted azacycloalkyl. R.sub.6 and R.sub.7 can
also form, together with the nitrogen atom to which they are
attached, a substituted or unsubstituted heterocyclic ring
structure.
In another embodiment of the compounds of Formula I, W, X, n,
R.sub.1, R.sub.3 and R.sub.4 have the meanings given above for
these variables; Z is --O--, --NR.sub.8--, alkylene-O-- or
alkylene-NR.sub.8--, where R.sub.8 is hydrogen or alkyl; and P is
hydrogen, alkyl, preferably normal or branched
C.sub.1-C.sub.4-alkyl or a peptide consisting of from 1 to about
100 amino acid residues attached at its carboxy terminus to Z.
In compounds of Formula I, when any of R.sub.1-R.sub.8 is an alkyl
group, preferred alkyl groups are substituted or unsubstituted
normal, branched or cyclic C.sub.1-C.sub.6 alkyl groups.
Particularly preferred alkyl groups are normal or branched
C.sub.1-C.sub.4 alkyl groups. A substituted alkyl group includes at
least one non-hydrogen substituent, such as an amino group, an
alkylamino group or a dialkylamino group; a halogen, such as a
fluoro, chloro, bromo or iodo substituent; or hydroxyl.
When at least one of R.sub.3 and R.sub.4 is a substituted or
unsubstituted aryl or heteroaryl group, preferred groups include
substituted and unsubstituted phenyl, naphthyl, indolyl, imidazolyl
and pyridyl. When at least one of R.sub.3 and R.sub.4 is
substituted or unsubstituted arylalkyl or heteroarylalkyl,
preferred groups include substituted and unsubstituted benzyl,
naphthylmethyl, indolylmethyl, imidazolylmethyl and pyridylmethyl
groups. Preferred substituents on aryl, heteroaryl, arylalkyl and
heteroarylalkyl groups are independently selected from the group
consisting of amino, alkyl-substituted amino, halogens, such as
fluoro, chloro, bromo and iodo; hydroxyl groups and alkyl groups,
preferably normal or branched C.sub.1-C.sub.6-alkyl groups, most
preferably methyl groups. X is preferably linear
C.sub.1-C.sub.6-alkylene, more preferably C.sub.1-C.sub.4-alkylene
and most preferably methylene or ethylene. When Z is
alkylene-C(O)--, alkylene-O-- or alkylene-NR.sub.8, the alkylene
group is preferably linear C.sub.1-C.sub.6-alkylene, more
preferably C.sub.1-C.sub.4-alkylene and most preferably methylene
or ethylene.
R.sub.6 and R.sub.7, in addition to alkyl, substituted alkyl or
hydrogen, can each also independently be a substituted or
unsubstituted azacycloalkyl group or a substituted or unsubstituted
azacycloalkylalkyl group. Suitable substituted azacycloalkyl groups
include azacycloalkyl groups which have an N-alkyl substituent,
preferably an N--C.sub.1-C.sub.4-alkyl substituent and more
preferably an N-methyl substituent. R.sub.6 and R.sub.7 can also,
together with the nitrogen atom to which they are attached, form a
heterocyclic ring system, such as a substituted or unsubstituted
five or six-membered aza- or diazacycloalkyl group. Preferably, the
diazacycloalkyl group includes an N-alkyl substituent, such as an
N--C.sub.1-C.sub.4-alkyl substituent or, more preferably, an
N-methyl substituent.
In particularly preferred embodiments, --N(R.sub.6)R.sub.7 is
NH.sub.2 or one of the groups shown below:
##STR00006##
Preferably, the compounds of Formula I do not include compounds
wherein Z is --O--, P is hydrogen, R.sub.3 and R.sub.4 are both
hydrogen, n is 1 and X is methylene. Preferably, the compounds of
Formula I further do not include compounds wherein Z is
methylene-O--, R.sub.3 and R.sub.4 are both hydrogen, and n is
0.
In another embodiment, the invention provides angiogenesis
inhibitor compounds of Formula XV,
##STR00007## where A is a MetAP-2 inhibitory core and W is O or NR.
In one embodiment, Z is --C(O)-- or -alkylene-C(O)-- and P is NHR,
OR or a peptide consisting of one to about one hundred amino acid
residues connected at the N-terminus to Z. In this embodiment, Q is
hydrogen, linear, branched or cyclic alkyl or aryl, provided that
when P is --OR, Q is not hydrogen. Z is preferably --C(O)-- or
C.sub.1-C.sub.4-alkylene-C(O)--, and, more preferably, --C(O)-- or
C.sub.1-C.sub.2-alkylene-C(O)--. Q is preferably linear, branched
or cyclic C.sub.1-C.sub.6-alkyl, phenyl or naphthyl. More
preferably, Q is isopropyl, phenyl or cyclohexyl.
In another embodiment, Z is -alkylene-O-- or -alkylene-N(R)--,
where alkylene is, preferably, C.sub.1-C.sub.6-alkylene, more
preferably C.sub.1-C.sub.4-alkylene and, most preferably,
C.sub.1-C.sub.2-alkylene. P is hydrogen or a peptide consisting of
from one to about one hundred amino acid residues connected to Z at
the carboxyl terminus. In this embodiment, Q is hydrogen, linear,
branched or cyclic alkyl or aryl, provided that when P is hydrogen,
Q is not hydrogen. Q is preferably linear, branched or cyclic
C.sub.1-C.sub.6-alkyl, phenyl or naphthyl. More preferably, Q is
isopropyl, phenyl or cyclohexyl.
In the compounds of Formula XV, each R is, independently, hydrogen
or alkyl. In one embodiment, each R is, independently, hydrogen or
linear, branched or cyclic C.sub.1-C.sub.6-alkyl. Preferably, each
R is, independently, hydrogen or linear or branched
C.sub.1-C.sub.4-alkyl. More preferably, each R is, independently,
hydrogen or methyl. In the most preferred embodiments, each R is
hydrogen.
In Formulas I and XV, A is a MetAP-2 inhibitory core. As used
herein, a "MetAP-2 inhibitory core" includes a moiety able to
inhibit the activity of methionine aminopeptidase 2 (MetAP-2),
e.g., the ability of MetAP-2 to cleave the N-terminal methionine
residue of newly synthesized proteins to produce the active form of
the protein. Preferred MetAP-2 inhibitory cores are Fumagillin
derived structures.
Suitable MetAP-2 inhibitory cores include the cores of Formula
II,
##STR00008## where R.sup.1 is hydrogen or alkoxy, preferably
C.sub.1-C.sub.4-alkoxy and more preferably, methoxy. R.sup.2 is
hydrogen or hydroxy; and R.sub.3 is hydrogen or alkyl, preferably
C.sub.1-C.sub.4-alkyl and more preferably, hydrogen. D is linear or
branched alkyl, preferably C.sub.1-C.sub.6-alkyl; arylalkyl,
preferably aryl-C.sub.1-C.sub.4-alkyl and more preferably
phenyl-C.sub.1-C.sub.4-alkyl; or D is of the structure
##STR00009## where the dashed line represents a single bond or a
double bond.
A can also be a MetAP-2 inhibitory core of Formula III,
##STR00010## Where R.sup.1, R.sup.2, R.sub.3 and D have the
meanings given above for Formula II, and X is a leaving group, such
as a halogen.
Examples of suitable MetAP-2 inhibitory cores include, but are not
limited to, the following.
##STR00011##
In each of Formulas IV-X, the indicated valence on the ring carbon
is the point of attachment of the structural variable W, as set
forth in Formulas I-XV. In Formula IX, p is an integer from 0 to
10, preferably 1-4. In Formulas IV, V and VI-IX, R.sub.1 is
hydrogen or C.sub.1-C.sub.4-alkoxy, preferably methoxy. In Formulas
IV and V, the dashed line indicates that the bond can be a double
bond or a single bond. In Formula V, X represents a leaving group,
such as a thioalkoxy group, a thioaryloxy group, a halogen or a
dialkylsulfinium group. In Formulas IV and V, R.sub.2 is H, OH,
amino, C.sub.1-C.sub.4-alkylamino or
di(C.sub.1-C.sub.4-alkyl)amino), preferably H. In formulas in which
the stereochemistry of a particular stereocenter is not indicated,
that stereocenter can have either of the possible
stereochemistries, consistent with the ability of the angiogenesis
inhibitor compound to inhibit the activity of MetAP-2.
In particularly preferred embodiments, A is the MetAP-2 inhibitory
core of Formula X below.
##STR00012##
As used herein, the terms "P" and "peptide" include compounds
comprising from 1 to about 100 amino acid residues (e.g., 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or
more amino acid residues). In preferred embodiments, the peptide
includes compounds comprising less than about 90, 80, 70, 60, 50,
40, 30, 20, or 10 amino acid residues, preferably about 1-10, 1-20,
1-30, 1-40, 1-50, 1-60, 1-70, 1-80, or 1-90 amino acid residues.
The peptides may be natural or synthetically made. The amino acid
residues are preferably .alpha.-amino acid residues. For example,
the amino acid residues can be independently selected from among
the twenty naturally occurring amino acid residues, the
D-enantiomers of the twenty natural amino acid residues, and may
also be non-natural amino acid residues (e.g., norleucine,
norvaline, phenylglycine, .beta.-alanine, or a peptide mimetic such
as 3-amino-methylbenzoic acid). In one embodiment, the amino acid
residues are independently selected from residues of Formula XI,
Formula XII, and Formula XIII.
##STR00013##
In Formula XI, X.sub.1 is hydrogen, a side chain of one of the
twenty naturally-occurring amino acid residues, a linear, branched
or cyclic C.sub.1-C.sub.8-alkyl group, an aryl group, such as a
phenyl or naphthyl group, an aryl-C.sub.1-C.sub.4-alkyl group, a
heteroaryl group, such as a pyridyl, thienyl, pyrrolyl, or furyl
group, or a heteroaryl-C.sub.1-C.sub.4-alkyl group; and X.sub.2 is
hydrogen a linear, branched or cyclic C.sub.1-C.sub.8-alkyl group,
an aryl group, such as a phenyl or naphthyl group, an
aryl-C.sub.1-C.sub.4-alkyl group or a heteroaryl group as described
above for X.sub.1. Preferably, X.sub.2 is hydrogen. In Formula XII,
Y is methylene, oxygen, sulfur or NH, and a and b are each,
independently, 0-4, provided that the sum of a and b is between 1
and 4. Formulas XI and XII encompass .alpha.-amino acid residues
having either a D or an L stereochemistry at the alpha carbon atom.
One or more of the amino acid residues can also be an amino acid
residue other than an .alpha.-amino acid residue, such as a
.beta.-, .gamma.- or .epsilon.-amino acid residue. Suitable
examples of such amino acid residues are of Formula XIII, wherein q
is an integer of from 2 to about 6, and each X.sub.1 and X.sub.2
independently have the meanings given above for these variables in
Formula XI.
In a preferred embodiment, the peptide used in the angiogenesis
inhibitor compounds of the invention may include a site-directed
sequence in order to increase the specificity of binding of the
angiogenesis inhibitor compound to a cell surface of interest. As
used herein, the term "site-directed sequence" is intended to
include any amino acid sequence (e.g., comprised of natural or non
natural amino acid residues) which serves to limit exposure of the
angiogenesis inhibitor compound to the periphery and/or which
serves to direct the angiogenesis inhibitor compound to a site of
interest, e.g., a site of angiogenesis or aberrant cellular
proliferation.
The peptide contained within the angiogenesis inhibitor compounds
of the invention may include a peptide cleavage site for an enzyme
which is expressed at sites of angiogenesis or aberrant cell
proliferation, allowing tissue-selective delivery of a
cell-permeable active angiogenesis inhibitor compound or fragment
thereof (e.g., a fragment containing the MetAP-2 inhibitory core of
the angiogenesis inhibitor compound). The peptide may also include
a sequence which is a ligand for a cell surface receptor which is
expressed at a site of angiogenesis or aberrant cell proliferation,
thereby targeting angiogenesis inhibitor compounds to a cell
surface of interest. For example, a peptide contained within the
angiogenesis inhibitor compounds of the invention can include a
cleavage site for a matrix metalloproteinase, or an integrin
binding RGD (Arg-Gly-Asp) sequence, or a combination of both an
enzyme "cleavage" sequence and a cell surface "ligand" which serve
to target the angiogenesis inhibitor compound to the membrane of an
endothelial cell. However, the selection of a peptide sequence must
be such that the active angiogenesis inhibitor compound is
available to be delivered to the cells in which MetAP-2 inhibition
is desired.
For example, a sequence that is cleaved by a matrix
matalloproteinase produces a product that contains the MetAP-2
inhibitory core, a coupling group, and a peptide fragment.
Sequences are selected so that the active angiogenesis inhibitor
compound, e.g., the active angiogenesis inhibitor compound
generated by the matrix matalloproteinase cleavage, is cell
permeable. Preferably, the active angiogenesis inhibitor compound
does not contain a free acid after the cleavage.
In one embodiment, the peptide includes a cleavage site for a
matrix metalloprotease, such as matrix metalloprotease-2 (MMP-2),
MMP-1, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MMP-13 or MMP-26.
Preferably, the peptide includes a cleavage site for MMP-2 or
MMP-9. For example, the peptide can comprise the sequence
-Pro-Leu-Gly-Xaa- (SEQ ID NO:1), where Xaa is any naturally
occurring amino acid residue consistent with matrix metalloprotease
(MMP) cleavage at the Gly-Xaa bond. Xaa is preferably a hydrophobic
amino acid residue, such as tryptophan, phenylalanine, methionine,
leucine, isoleucine, proline, and valine.
Other suitable sequences include sequences comprising one or more
of Pro-Cha-Gly-Cys(Me)-His (SEQ ID NO:2);
Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (SEQ ID NO:3);
Pro-Gln-Gly-Ile-Ala-Gly-Trp (SEQ ID NO:4);
Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg (SEQ ID NO:5);
Pro-Leu-Gly-Met-Trp-Ser-Arg (SEQ ID NO:35);
Pro-Leu-Gly-Leu-Trp-Ala-D-Arg (SEQ ID NO:6);
Pro-Leu-Ala-Leu-Trp-Ala-Arg (SEQ ID NO:7);
Pro-Leu-Ala-Leu-Trp-Ala-Arg (SEQ ID NO:8);
Pro-Leu-Ala-Tyr-Trp-Ala-Arg (SEQ ID NO:9);
Pro-Tyr-Ala-Tyr-Trp-Met-Arg (SEQ ID NO:10); Pro-Cha-Gly-Nva-His-Ala
(SEQ ID NO:11); Pro-Leu-Ala-Nva (SEQ ID NO:12); Pro-Leu-Gly-Leu
(SEQ ID NO:13); Pro-Leu-Gly-Ala (SEQ ID NO:14);
Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser (SEQ ID NO:15);
Pro-Cha-Ala-Abu-Cys(Me)-His-Ala (SEQ ID NO:16);
Pro-Cha-Ala-Gly-Cys(Me)-His-Ala (SEQ ID NO:17);
Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu (SEQ ID NO:18);
Pro-Lys-Pro-Leu-Ala-Leu (SEQ ID NO:19);
Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met (SEQ ID NO:20);
Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg (SEQ ID NO:21);
Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg (SEQ ID NO:22); and
Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp (SEQ ID NO:23). These sequences
identify the natural amino acid residues using the customary
three-letter abbreviations; the following abbreviations represent
the indicated non-natural amino acids: Abu=L-a-aminobutyryl;
Cha=L-cyclohexylalanine; Nva=L-norvaline.
In certain embodiments, P is an amino acid sequence selected from
the group consisting of Ac-Pro-Leu-Gly-Met-Trp-Ala (SEQ ID NO:24);
Gly-Pro-Leu-Gly-Met-His-Ala-Gly (SEQ ID NO:25); Gly-Pro-Leu-(Me)Gly
(SEQ ID NO:26); Gly-Pro-Leu-Gly (SEQ ID NO:27); Gly-Met-Gly-Leu-Pro
(SEQ ID NO:28); Ala-Met-Gly-Ile-Pro (SEQ ID NO:29);
Gly-Arg-Gly-Asp-(O-Me-Tyr)-Arg-Glu (SEQ ID NO:30);
Gly-Arg-Gly-Asp-Ser-Pro (SEQ ID NO:31); Gly-Arg-Gly-Asp (SEQ ID
NO:32); Asp-Gly-Arg; Ac-Pro-Leu-Gly-Met-Ala (SEQ ID NO:34);
Ac-Arg-Gly-Asp-Ser-Pro-Leu-Gly-Met-Trp-Ala (SEQ ID NO:33);
Ac-Pro-Leu-Gly-Met-Gly (SEQ ID NO:36); Met-Trp-Ala (SEQ ID NO:37);
Met-Gly (SEQ ID NO:38); Gly-Pro-Leu-Gly-Met-Trp-Ala-Gly (SEQ ID
NO:39); and Gly-Arg-Gly-(3-amino-3-pyridylpropionic acid) (SEQ ID
NO:40). (Ac in the foregoing sequences represents an Acetyl
group).
The peptide can be attached to the MetAP-2 inhibitory core at
either its N-terminus or C-terminus. When the peptide is attached
to the MetAP-2 inhibitory core at its C-terminus, the N-terminus of
the peptide can be --NR.sub.2R.sub.3, where R.sub.2 is hydrogen,
alkyl or arylalkyl and R.sub.3 is hydrogen, alkyl, arylalkyl or
acyl. When the peptide is attached to the MetAP-2 inhibitory core
at its N-terminus, the C-terminus can be --C(O)R.sub.4, where
R.sub.4 is --OH, --O-alkyl, --O-arylalkyl, or --NR.sub.2R.sub.3,
where R.sub.2 is hydrogen, alkyl or arylalkyl and R.sub.3 is
hydrogen, alkyl, arylalkyl or acyl. In this embodiment, the
C-terminal residue can also be present in a reduced form, such as
the corresponding primary alcohol.
The present invention also includes pharmaceutically acceptable
salts of the angiogenesis inhibitor compounds of the invention. A
"pharmaceutically acceptable salt" includes a salt that retains the
desired biological activity of the parent angiogenesis inhibitor
compound and does not impart any undesired toxicological effects.
Examples of such salts are salts of acids such as hydrochloric
acid, hydrobromic acid, sulfuric acid, phosporic acid, nitric acid,
and the like; acetic acid, oxalic acid, tartaric acid, succinic
acid, malic acid, benzoic acid, pamoic acid, alginic acid,
methanesulfonic acid, naphthalenesulfonic acid, and the like. Also
included are salts of cations such as sodium, potassium, lithium,
zinc, copper, barium, bismuth, calcium, and the like; or organic
cations such as trialkylammonium. Combinations of the above salts
are also useful.
Preferred Angiogenesis Inhibitor Compounds of Formula I
One set of particularly preferred angiogenesis inhibitor compounds
of the invention includes compounds in which A is the MetAP-2
inhibitory core of Formula X, W is O or NR.sub.2, and the
structure
##STR00014## is represented by the structures set forth below.
##STR00015## ##STR00016## ##STR00017## ##STR00018## ##STR00019##
##STR00020## ##STR00021## ##STR00022## ##STR00023## ##STR00024##
##STR00025## ##STR00026## Preferred Angiogenesis Inhibitor
Compounds of Formula XV
A preferred subset of the angiogenesis inhibitor compounds of
Formula XV comprises Formula XIV shown below.
##STR00027##
In one embodiment, W is O or NR. Z is --C(O) or -alkylene-C(O)--,
preferably C.sub.1-C.sub.4-alkylene-C(O)--. R is hydrogen or a
C.sub.1-C.sub.4-alkyl. Q is hydrogen; linear, branched or cyclic
C.sub.1-C.sub.6-alkyl; or aryl. R.sub.1 is hydroxy,
C.sub.1-C.sub.4-alkoxy or halogen. P is NH.sub.2, OR or a peptide
attached to Z at its N-terminus and comprising from 1 to 100 amino
acid residues independently selected from naturally occurring amino
acid residues, D-enantiomers of the naturally occurring amino acid
residues and non-natural amino acid residues. When Q is H, P is not
NH.sub.2 or OR. In preferred embodiments, W is O or NH; Q is
isopropyl; R.sub.1 is methoxy; P comprises from 1 to 15 amino acid
residues; and the dashed line present in Formula XIV represents a
double bond. In particularly preferred embodiments, W is O, and P
comprises 10 or fewer amino acid residues.
In another embodiment of the compounds of Formula XIV, W is O or
NR. Z is alkylene-O or alkylene-NR, preferably C1-C4-alkylene-O or
C1-C4-alkylene-NR--. R is hydrogen or a C.sub.1-C.sub.4-alkyl. Q is
hydrogen; linear, branched or cyclic C.sub.1-C.sub.6-alkyl; or
aryl. R.sub.1 is hydroxy, C.sub.1-C.sub.4-alkoxy or halogen. P is
hydrogen or a peptide attached to Z at its C-terminus and
comprising from 1 to 100 amino acid residues independently selected
from naturally occurring amino acid residues, D-enantiomers of the
naturally occurring amino acid residues and non-natural amino acid
residues. When Q is H, P is not H. In preferred embodiments, W is O
or NH; Q is isopropyl; R.sub.1 is methoxy; P comprises from 1 to 15
amino acid residues; and the dashed line present in Formula XIV
represents a double bond. In particularly preferred embodiments, W
is O, and P comprises 10 or fewer amino acid residues or P is
hydrogen.
One set of particularly preferred angiogenesis inhibitor compounds
of the invention is represented by the structures set forth
below.
##STR00028## ##STR00029## ##STR00030## ##STR00031## ##STR00032##
##STR00033## Methods of Using the Angiogenesis Inhibitor Compounds
for the Treatment of Angiogenic Disease
In another embodiment, the present invention provides a method of
treating an angiogenic disease in a subject. The method includes
administering to the subject a therapeutically effective amount of
an angiogenesis inhibitor compound of the present invention,
thereby treating the angiogenic disease in the subject.
As used herein, the term "angiogenic disease" includes a disease,
disorder, or condition characterized or caused by aberrant or
unwanted, e.g., stimulated or suppressed, formation of blood
vessels (angiogenesis). Aberrant or unwanted angiogenesis may
either cause a particular disease directly or exacerbate an
existing pathological condition. Examples of angiogenic diseases
include ocular disorders, e.g., diabetic retinopathy, retinopathy
of prematurity, corneal graft rejection, retrolental fibroplasia,
neovascular glaucoma, rubeosis, retinal neovascularization due to
macular degeneration, hypoxia, angiogenesis in the eye associated
with infection or surgical intervention, ocular tumors and
trachoma, and other abnormal neovascularization conditions of the
eye, where neovascularization may lead to blindness; disorders
affecting the skin, e.g., psoriasis and pyogenic granuloma; cancer,
e.g., carcinomas and sarcomas, where progressive growth is
dependent upon the continuous induction of angiogenesis by these
tumor cells, lung cancer, brain cancer, kidney cancer, colon
cancer, liver cancer, pancreatic cancer, stomach cancer, prostate
cancer, breast cancer, ovarian cancer, cervical cancer, melanoma,
and metastatic versions of any of the preceding cancers; lymphoid
malignancies, e.g., lymphoid leukemias, such as chronic lymphoid
leukemia and acute lymphoid leukemia, and lymphomas, such as T cell
lymphoma and B cell lymphoma; pediatric disorders, e.g.,
angiofibroma, and hemophiliac joints; blood vessel diseases such as
hemangiomas, and capillary proliferation within atherosclerotic
plaques; disorders associated with surgery, e.g., hypertrophic
scars, wound granulation and vascular adhesions; and autoimmune
diseases such as rheumatoid, immune and degenerative arthritis,
where new vessels in the joint may destroy articular cartilage and
scleroderma; lupus erythematosus, psoriasis, multiple sclerosis,
myasthenia gravis, vasculitis, or diabetes mellitus.
The term angiogenic disease also includes diseases characterized by
excessive or abnormal stimulation of endothelial cells, including
but not limited to intestinal adhesions, Crohn's disease,
atherosclerosis, scleroderma, and hypertrophic scars, i.e.,
keloids; diseases that have angiogenesis as a pathologic
consequence such as cat scratch disease (Rochele ninalia quintosa)
and ulcers (Helicobacter pylori). In addition, the angiogenesis
inhibitor compounds of the present invention are useful as birth
control agents (by virtue of their ability to inhibit the
angiogenesis dependent ovulation and establishment of the placenta)
and may also be used to reduce bleeding by administration to a
subject prior to surgery.
The compounds of the invention may also be used to treat a subject
suffering from a parasitic infection, such as an infection by
Plasmodium species, such as Plasmodium falciparum, or an infection
by Leishmania species, such as Leishmania donavani. The method
comprises the step of administering to the subject a
therapeutically effective amount of a compound of the invention.
The subject can be an individual who is suffering from, or
susceptible to, infection by a parasitic organism. In a preferred
embodiment, the subject suffers from malaria or Leishmaniasis.
The compounds of the invention can also be used to treat a subject
suffering from a thymoma. Thus the invention provides a method of
treating a thymoma in a patient, comprising the step of
administering to the patient a therapeutically effective amount of
a compound of the invention.
The compounds of the invention can also be used as
immunosuppressive agents in clinical protocols in which suppression
of the immune system is desired. Thus, the present invention
provides a method of inducing an immunosupressed condition in a
subject, comprising the step of administering to the subject an
immunosupressive amount of a compound of the invention. For
example, the compounds of the invention can be used to suppress
immune function in subjects undergoing, or who have undergone, an
organ, tissue or cell transplant from a donor. In one embodiment,
the transplanted tissue, organ or cell is bone marrow, stem cells,
pancreatic cells, such as islet cells, or cornea. In another
embodiment, the transplanted organ is a solid organ, such as a
liver, a kidney, a heart or a lung.
The compounds of the invention may also be used to treat a subject
(e.g., a mammal, such as a human) suffering from a lymphoid
malignancy. The method includes administering to a subject an
effective amount of a MetAP-2 inhibitor, thereby treating a subject
suffering from a lymphoid malignancy.
The compounds of the invention may also be used to treat rheumatic
diseases, such as rheumatoid arthritis, lupus, akylosing
spondylitis, psoriatic arthritis, scleroderma, Kawasaki syndrome
and other rheumatic diseases as set forth in Primer on the
Rheumatic Diseases, 11th Edition (John H. Klippel, MD, editor;
Arthritis Foundation: Atlanta Ga. (1997)).
As used herein, the term "lymphoid malignancy" includes any
malignancy of a lymphoid cell. Examples of lymphoid malignancies
include lymphoid leukemias, such as chronic lymphoid leukemia and
acute lymphoid leukemia, and lymphomas, such as Non-Hogkins
lymphoma. The term "Non-Hodgkins lymphoma" includes T cell
lymphomas, such as Precursor (peripheral) T-cell lymphoblastic,
Adult T-cell, extranodal Natural Killer/T-cell, nasal type,
enteropathy type T-cell, hepatosplenic T-cell, subcutaneous
panniculitis like T-cell, skin (cutaneous) lymphomas, anaplastic
large cell, peripheral T-cell, and angioimmunoblastic T-cell
lymphomas; and B cell lymphomas, such as precursor B lymphoblastic,
small lymphocytic, B-cell prolymphocytic, lymphoplasmacytic,
splenic marginal zone, extranodal marginal zone--MALT, nodal
marginal zone, follicular, mantle cell, diffuse large B-cell,
primary mediastinal large B-cell, primary effusion and Burkitt's
lymphomas. Non-Hodgkins lymphoma also includes AIDS-related
lymphoma and central nervous system lymphoma.
As used herein, the term "subject" includes warm-blooded animals,
preferably mammals, including humans. In a preferred embodiment,
the subject is a primate. In an even more preferred embodiment, the
primate is a human.
As used herein, the term "administering" to a subject includes
dispensing, delivering or applying an angiogenesis inhibitor
compound, e.g., an angiogenesis inhibitor compound in a
pharmaceutical formulation (as described herein), to a subject by
any suitable route for delivery of the compound to the desired
location in the subject, including delivery by either the
parenteral or oral route, intramuscular injection,
subcutaneous/intradermal injection, intravenous injection, buccal
administration, transdermal delivery and administration by the
rectal, colonic, vaginal, intranasal or respiratory tract
route.
As used herein, the term "effective amount" includes an amount
effective, at dosages and for periods of time necessary, to achieve
the desired result, e.g., sufficient to treat an angiogenic disease
in a subject. An effective amount of an angiogenesis inhibitor
compound, as defined herein may vary according to factors such as
the disease state, age, and weight of the subject, and the ability
of the angiogenesis inhibitor compound to elicit a desired response
in the subject. Dosage regimens may be adjusted to provide the
optimum therapeutic response. An effective amount is also one in
which any toxic or detrimental effects (e.g., side effects) of the
angiogenesis inhibitor compound are outweighed by the
therapeutically beneficial effects.
A therapeutically effective amount of an angiogenesis inhibitor
compound (i.e., an effective dosage) may range from about 0.001 to
30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body
weight, more preferably about 0.1 to 20 mg/kg body weight, and even
more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4
to 7 mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will
appreciate that certain factors may influence the dosage required
to effectively treat a subject, including but not limited to the
severity of the disease or disorder, previous treatments, the
general health and/or age of the subject, and other diseases
present. Moreover, treatment of a subject with a therapeutically
effective amount of an angiogenesis inhibitor compound can include
a single treatment or, preferably, can include a series of
treatments. In one example, a subject is treated with an
angiogenesis inhibitor compound in the range of between about 0.1
to 20 mg/kg body weight, one time per week for between about 1 to
10 weeks, preferably between 2 to 8 weeks, more preferably between
about 3 to 7 weeks, and even more preferably for about 4, 5, or 6
weeks. It will also be appreciated that the effective dosage of an
angiogenesis inhibitor compound used for treatment may increase or
decrease over the course of a particular treatment.
The methods of the invention further include administering to a
subject a therapeutically effective amount of an angiogenesis
inhibitor compound in combination with another pharmaceutically
active compound known to treat an angiogenic disease, e.g., a
chemotherapeutic agent such as Taxol, Paclitaxel, or Actinomycin D,
or an antidiabetic agent such as Tolbutamide; or a compound that
may potentiate the angiogenesis inhibitory activity of the
angiogenesis inhibitor compound, such as heparin or a sulfated
cyclodextrin. Other pharmaceutically active compounds that may be
used can be found in Harrison's Principles of Internal Medicine,
Thirteenth Edition, Eds. T. R. Harrison et al. McGraw-Hill N.Y.,
N.Y.; and the Physicians Desk Reference 50th Edition 1997, Oradell
N.J., Medical Economics Co., the complete contents of which are
expressly incorporated herein by reference. The angiogenesis
inhibitor compound and the pharmaceutically active compound may be
administered to the subject in the same pharmaceutical composition
or in different pharmaceutical compositions (at the same time or at
different times).
Pharmaceutical Compositions of the Angiogenesis Inhibitor
Compounds
The present invention also provides pharmaceutically acceptable
formulations comprising one or more angiogenesis inhibitor
compounds. Such pharmaceutically acceptable formulations typically
include one or more angiogenesis inhibitor compounds as well as a
pharmaceutically acceptable carrier(s) and/or excipient(s). As used
herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and anti fungal
agents, isotonic and absorption delaying agents, and the like that
are physiologically compatible. The use of such media and agents
for pharmaceutically active substances is well known in the art.
Except insofar as any conventional media or agent is incompatible
with the angiogenesis inhibitor compounds, use thereof in the
pharmaceutical compositions is contemplated.
Supplementary pharmaceutically active compounds known to treat an
angiogenic disease, e.g., a chemotherapeutic agent such as Taxol,
Paclitaxel, or Actinomycin D, or an antidiabetic agent such as
Tolbutamide; or compounds that may potentiate the angiogenesis
inhibitory activity of the angiogenesis inhibitor compound, such as
heparin or a sulfated cyclodextrin, can also be incorporated into
the compositions of the invention. Suitable pharmaceutically active
compounds that may be used can be found in Harrison's Principles of
Internal Medicine (supra).
A pharmaceutical composition of the invention is formulated to be
compatible with its intended route of administration. Examples of
routes of administration include parenteral, e.g., intravenous,
intradermal, subcutaneous, oral (e.g., inhalation), transdermal
(topical), transmucosal, and rectal administration. Solutions or
suspensions used for parenteral, intradermal, or subcutaneous
application can include the following components: a sterile diluent
such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
Pharmaceutical compositions suitable for injectable use include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersion. For intravenous administration,
suitable carriers include physiological saline, bacteriostatic
water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate
buffered saline (PBS). In all cases, the pharmaceutical composition
must be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
Sterile injectable solutions can be prepared by incorporating the
angiogenesis inhibitor compound in the required amount in an
appropriate solvent with one or a combination of the ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the
angiogenesis inhibitor compound into a sterile vehicle which
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying which yields a powder of the angiogenesis inhibitor
compound plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible
carrier. They can be enclosed in gelatin capsules or compressed
into tablets. For the purpose of oral therapeutic administration,
the angiogenesis inhibitor compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also include an enteric coating. Oral
compositions can also be prepared using a fluid carrier for use as
a mouthwash, wherein the angiogenesis inhibitor compound in the
fluid carrier is applied orally and swished and expectorated or
swallowed. Pharmaceutically compatible binding agents, and/or
adjuvant materials can be included as part of the composition. The
tablets, pills, capsules, troches and the like can contain any of
the following ingredients, or compounds of a similar nature: a
binder such as microcrystalline cellulose, gum tragacanth or
gelatin; an excipient such as starch or lactose, a disintegrating
agent such as alginic acid, Primogel, or corn starch; a lubricant
such as magnesium stearate or Sterotes; a glidant such as colloidal
silicon dioxide; a sweetening agent such as sucrose or saccharin;
or a flavoring agent such as peppermint, methyl salicylate, or
orange flavoring.
For administration by inhalation, the angiogenesis inhibitor
compounds are delivered in the form of an aerosol spray from
pressured container or dispenser which contains a suitable
propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal
means. For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art, and
include, for example, for transmucosal administration, detergents,
bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the angiogenesis
inhibitor compounds are formulated into ointments, salves, gels, or
creams as generally known in the art.
The angiogenesis inhibitor compounds can also be prepared in the
form of suppositories (e.g., with conventional suppository bases
such as cocoa butter and other glycerides) or retention enemas for
rectal delivery.
In one embodiment, the angiogenesis inhibitor compounds are
prepared with carriers that will protect the compound against rapid
elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions can also be used as pharmaceutically acceptable
carriers. These can be prepared according to methods known to those
skilled in the art, for example, as described in U.S. Pat. Nos.
4,522,811, 5,455,044 and 5,576,018, and 4,883,666, the contents of
all of which are incorporated herein by reference.
The angiogenesis inhibitor compounds of the invention can also be
incorporated into pharmaceutical compositions which allow for the
sustained delivery of the angiogenesis inhibitor compounds to a
subject for a period of at least several weeks to a month or more.
Such formulations are described in U.S. Pat. No. 5,968,895, the
contents of which are incorporated herein by reference.
It is especially advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of
angiogenesis inhibitor compounds calculated to produce the desired
therapeutic effect in association with the required pharmaceutical
carrier. The specification for the dosage unit forms of the
invention are dictated by and directly dependent on the unique
characteristics of the angiogenesis inhibitor compound and the
particular therapeutic effect to be achieved, and the limitations
inherent in the art of compounding such angiogenesis inhibitor
compounds for the treatment of individuals.
Toxicity and therapeutic efficacy of such angiogenesis inhibitor
compounds can be determined by standard pharmaceutical procedures
in cell cultures or experimental animals, e.g., for determining the
LD50 (the dose lethal to 50% of the population) and the ED50 (the
dose therapeutically effective in 50% of the population). The dose
ratio between toxic and therapeutic effects is the therapeutic
index and it can be expressed as the ratio LD50/ED50. Angiogenesis
inhibitor compounds which exhibit large therapeutic indices are
preferred. While angiogenesis inhibitor compounds that exhibit
toxic side effects may be used, care should be taken to design a
delivery system that targets such angiogenesis inhibitor compounds
to the site of affected tissue in order to minimize potential
damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies
can be used in formulating a range of dosage for use in humans. The
dosage of such angiogenesis inhibitor compounds lies preferably
within a range of circulating concentrations that include the ED50
with little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any angiogenesis inhibitor compounds
used in the methods of the invention, the therapeutically effective
dose can be estimated initially from cell culture assays. A dose
may be formulated in animal models to achieve a circulating plasma
concentration range that includes the IC50 (i.e., the concentration
of the angiogenesis inhibitor compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.
Assays for Detecting the Activity of the Angiogenesis Inhibitor
Compounds
The angiogenesis inhibitor compounds of the invention may be tested
for their ability to modulate (e.g., inhibit or stimulate)
angiogenesis in a variety of well known assays, e.g., the rat
aortic ring angiogenesis inhibition assay or in a chorioallantoic
membrane (CAM) assay.
The CAM assay may be performed essentially as described in Liekens
S. et al. (1997) Oncology Research 9: 173-181, the contents of
which are incorporated herein by reference. Briefly, fresh
fertilized eggs are incubated for 3 days at 37.degree. C. On the
third day, the shell is cracked and the egg is placed into a tissue
culture plate and incubated at 38.degree. C. For the assay, the
angiogenesis inhibitor compound to be tested is attached on a
matrix of collagen on a nylon mesh. The mesh is then used to cover
the chorioallantoic membrane and the eggs are incubated at
37.degree. C. If angiogenesis occurs, new capillaries form and grow
through the mesh within 24 hours. The ability of the angiogenesis
inhibitor compound (at various concentrations) to modulate, e.g.,
inhibit, angiogenesis, e.g., FGF-induced angiogenesis, may then be
determined.
The angiogenesis inhibitor compounds of the invention may also be
tested for their ability to modulate (e.g., inhibit or stimulate)
human endothelial cell growth. Human umbilical vein endothelial
cells (HUVE) may be isolated by perfusion of an umbilical vein with
a trypsin-containing medium. HUVE may then be cultured in GIT
medium (Diago Eiyou Kagaku, Co., Japan) supplemented with 2.5%
fetal bovine serum and 2.0 ng/ml of recombinant human basic
fibroblast growth factor (rbFGF, Biotechnology Research
Laboratories, Takeda, Osaka, Japan) at 37.degree. C. under 5%
CO.sub.2 and 7% O.sub.2. HUVE are then plated on 96-well microtiter
plates (Nunc, 1-67008) at a cell density of 2.times.10.sup.3/100
.mu.l of medium. The following day, 100 .mu.l of medium containing
rbFGF (2 ng/ml at the final concentration) and each angiogenesis
inhibitor compound at various concentrations may be added to each
well. The angiogenesis inhibitor compounds are dissolved in
dimethylsulfoxide (DMSO) and then diluted with culture medium so
that the final DMSO concentration does not exceed 0.25%. After a
5-day culture, medium is removed, 100 .mu.l of 1 mg/ml of MTT
(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide)
solution is added to the wells, and microtiters are kept at
37.degree. C. for 4 hours. Then, 100 .mu.l of 10% sodium dodecyl
sulfate (SDS) solution is added to wells, and the microtiters are
kept at 37.degree. C. for 5-6 hours. To determine the effects of
the angiogenesis inhibitor compound on cell number, the optical
density (590 .mu.m) of each well is measured using an optical
densitometer.
The ability of the angiogenesis inhibitor compounds of the
invention to modulate capillary endothelial cell migration in vitro
may also be tested using the Boyden chamber assay (as described in
Falk et al. (1980) J. Immunol. Meth. 33:239-247, the contents of
which are incorporated herein by reference). Briefly, bovine
capillary endothelial cells are plated at 1.5.times.10.sup.4 cells
per well in serum-free DMEM (Dulbecco's Modified Eagle's Medium) on
one side of nucleopore filters pre-coated with fibronectin (7.3
.mu.g fibronectin/ml PBS). An angiogenesis inhibitor compound is
dissolved in ethanol and diluted in DMEM so that the final
concentration of ethanol does not exceed 0.01%. Cells are exposed
to endothelial mitogen (Biomedical Technologies, Mass.) at 200
.mu.g/ml and different concentrations of the angiogenesis inhibitor
compound in serum-free DMEM for 4 hours at 37.degree. C. At the end
of this incubation, the number of cells that migrate through 8.mu.
pores in the filters is determined by counting cells with an ocular
grid at 100.times. in quadruplicate.
The ability of the angiogenesis inhibitor compounds of the
invention to modulate tumor growth may be tested in vivo. An animal
model, e.g., a C57BL/6N mouse with a mouse reticulum cell sarcoma
(M 5076) intraperitoneally transplantated therein, may be used. The
tumor cells in ascites can be collected by centrifugation, and
suspended in saline. The cell suspension (2.times.10.sup.6
cells/100 .mu.l/mouse) is inoculated into the right flanks of mice.
Tumor-bearing mice are then subcutaneously treated with the
angiogenesis inhibitor compound (at various concentrations
suspended in 5% arabic gum solution containing 1% of ethanol) for
12 days beginning one day after the tumor inoculation. The tumor
growth may be determined by measuring tumor size in two directions
with calipers at intervals of a few days.
Finally, the ability of the angiogenesis inhibitor compounds of the
invention to modulate the activity of MetAP2 may be tested as
follows. Recombinant human MetAP2 may be expressed and purified
from insect cells as described in Li and Chang, (1996) Biochem.
Biophys. Res. Commun. 227:152-159. Various amounts of angiogenesis
inhibitor compound is then added to buffer H (10 mM Hepes, pH 7.35,
100 mM KCl, 10% glycerol, and 0.1 M Co.sup.2+) containing InM
purified recombinant human MetAP2 and incubated at 37.degree. C.
for 30 minutes. To start the enzymatic reaction a peptide
containing a methionine residue, e.g., Met-Gly-Met, is added to the
reaction mixture (to a concentration of 1 mM). Released methionine
is subsequently quantified at different time points (e.g., at 0, 2,
3, and 5 minutes) using the method of Zou et al. (1995) Mol. Gen
Genetics 246:247-253).
This invention is further illustrated by the following examples
which should not be construed as limiting. The contents of all
references, patents and published patent applications cited
throughout this application, as well as the Figures and the
Sequence Listing, are hereby incorporated by reference.
EXAMPLES
Synthetic Methods
Compounds of the invention can be prepared using one or more of the
following general methods.
General Procedure A: To a mixture of carbonic acid-(3R, 4S, 5S,
6R)-5-methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yl
ester 4-nitro-phenyl ester.sup.1 (1, 0.47 mmol; Han, C. K.; Ahn, S.
K.; Choi, N. S.; Hong, R. K.; Moon, S. K.; Chun, H. S.; Lee, S. J.;
Kim, J. W.; Hong, C. I.; Kim, D.; Yoon, J. H.; No, K. T. Biorg.
Med. Chem. Lett. 2000, 10, 39-43) and amine (2.35 mmol) in EtOH (9
mL) was added dropwise, diisopropyl ethyl amine (2.35 mmol). After
3-18 hours, the ethanol was removed in vacuo and the crude material
was dissolved into EtOAc (10 mL) and washed with H.sub.2O
(2.times.5 mL), and then brine (5 mL). The organic phase was dried
over Na.sub.2SO.sub.4 and the solvent removed in vacuo.
Purification via flash chromatography (2-5% MeOH/CH.sub.2Cl.sub.2)
afforded product.
General Procedure B, Part I: A solution of (3R, 4S, 5S,
6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylox-
ycarbonylamino)-acetic acid.sup.2 (2, 0.11 mmol; U.S. Pat. No.
6,017,954) in DMF (1 mL) was added to a 10 mL round bottomed flask
containing swelled PS-DCC (0.28 mmol). In a separate vessel, the
peptide (0.04 mmol) was dissolved into DMF (0.5 mL) and neutralized
with NMM (0.04 mmol). After 1 hour, the solution of peptide was
added to the pre-activated acid, and the reaction was continued for
5-18 hours. The resin was removed by filtration, washed with DMF
(0.5 mL) and the solvent removed in vacuo. Purification via HPLC
(CH.sub.3CN/H.sub.2O) afforded the product.
General Procedure B, Part II: A solution of the product in Part I
(0.009 mmol) was dissolved into MeOH (1 mL) and was treated with
Pd/C (2 mg), then subjected to a H.sub.2 atmosphere (38 psi) for 24
hours. The mixture was then filtered through Celite, washed with
MeOH (0.5 mL) and the solvent removed in vacuo. Purification via
HPLC (CH.sub.3CN/H.sub.2O) afforded the product as a white
solid.
General Procedure C: (1-Hydroxymethyl-methyl-propyl)-carbamic acid
(3R, 4S, 5S, 6R)-5-methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yl
ester (Example7, 189 mg, 0.46 mmol), acid (0.46 mmol) and DMAP
(0.69 mmol) were dissolved into anhydrous CH.sub.2Cl.sub.2 (5 mL)
and treated with diisopropylcarbodiimide (0.46 mmol). After 7-18
hours, the solvent was removed in vacuo and purification via flash
chromatography (MeOH/CH.sub.2Cl.sub.2) afforded the product.
Example 1
2-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylox-
ycarbonylamino}-3-methyl-butyric acid methyl ester
##STR00034##
General procedure A was followed using 1 (31 mg, 0.07 mmol),
L-valine methyl ester hydrochloride (58 mg, 0.35 mmol), and DIEA
(60 .mu.L, 0.35 mmol) in EtOH (2 mL). Purification via flash
chromatography (1% MeOH/CH.sub.2Cl.sub.2) afforded the product as a
clear oil (10 mg, 0.02 mmol, 33% yield); R.sub.f=0.60 (20%
EtOAc/CH.sub.2Cl.sub.2); LRMS (m/z) [M+1].sup.+ 440.3 (calculated
for C.sub.23H.sub.38NO.sub.7, 440.3).
Example 2
2-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylox-
ycarbonylamino}-3-methyl-butyric acid methyl ester
##STR00035##
General procedure A was followed using 1 (41 mg, 0.09 mmol) and
D-valine methyl ester hydrochloride (77 mg, 0.45 mmol), and DIEA
(80 .mu.L, 0.45 mmol) in EtOH (2 mL). Purification via flash
chromatography (1% MeOH/CH.sub.2Cl.sub.2) afforded the product as a
clear oil (18 mg, 0.04 mmol, 45% yield); R.sub.f=0.39 (20%
EtOAc/CH.sub.2Cl.sub.2; LRMS (m/z) [M+1].sup.+ 440.3 (calculated
for C.sub.23H.sub.38NO.sub.7, 440.3).
Example 3
2-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylox-
ycarbonylamino}-4-methyl-pentanoic acid methyl ester
##STR00036##
General procedure A was followed using 1 (23 mg, 0.05 mmol),
D-leucine methyl ester hydrochloride (47 mg, 0.25 mmol), and DIEA
(45 .mu.L, 0.25 mmol) in EtOH (2 mL). Purification via flash
chromatography (1% MeOH/CH.sub.2Cl.sub.2) afforded the product as a
clear oil (19 mg, 0.04 mmol, 83% yield); R.sub.f=0.22 (15%
EtOAc/CH.sub.2Cl.sub.2); LRMS (m/z) [M+1].sup.+ 454.3 (calculated
for C.sub.24H.sub.40NO.sub.7, 454.3).
Example 4
{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylox-
ycarbonylamino}-phenyl-acetic acid methyl ester
##STR00037##
General procedure A was followed using 1 (37 mg, 0.08 mmol),
D-phenyl glycine methyl ester hydrochloride (83 mg, 0.40 mmol), and
DIEA (72 .mu.L, 0.40 mmol) in EtOH (2 mL). Purification via flash
chromatography (1% MeOH/CH.sub.2Cl.sub.2) afforded the product as a
clear oil (32 mg, 0.07 mmol, 82% yield); R.sub.f=0.41 (2%
MeOH/CH.sub.2Cl.sub.2); LRMS (m/z) [M+1].sup.+ 474.3 (calculated
for C.sub.26H.sub.36NO.sub.7, 474.3).
Example 5
(1-Carbamoyl-2-methyl-propyl)-carbamic acid-(3R, 4S, 5S,
6R)-5-methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yl
ester
##STR00038##
General procedure A was followed using 1 (55 mg, 0.12 mmol),
D-valine amide hydrochloride (93 mg, 0.62 mmol), and DIEA (110
.mu.L, 0.62 mmol) in EtOH (2 mL). Purification via flash
chromatography (2% MeOH/CH.sub.2Cl.sub.2) afforded the product as a
clear oil (42 mg, 0.10 mmol, 80% yield); R.sub.f=0.19 (2%
MeOH/CH.sub.2Cl.sub.2); LRMS (m/z) [M+1].sup.+ 425.5 (calculated
for C.sub.22H.sub.37N.sub.2O.sub.6, 425.5).
Example 6
(1-Carbamoyl-2-methyl-propyl)-carbamic acid-(3R, 4S, 5S,
6R)-5-methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-butyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yl
ester
##STR00039##
The compound in Example 4 (18 mg, 0.04 mmol) was dissolved into
anhydrous MeOH (1.5 mL) and treated with Pd--C (2 mg) under a
H.sub.2 atmosphere. After 12 hours, the reaction was filtered
through Celite and the solvent removed in vacuo to afford the
product as a clear oil (18 mg, 0.04 mmol, 100% yield); R.sup.f=0.21
(2% MeOH/CH.sub.2Cl.sub.2); LRMS (m/z) [M+1].sup.+ 427.5
(calculated for C.sub.22H.sub.39N.sub.2O.sub.6, 427.5).
Example 7
(1-Hydroxymethyl-2-methyl-propyl)-carbamic acid-(3R, 4S, 5S,
6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-
-spiro[2.5]oct-6-yl ester
##STR00040##
General procedure A was followed using 1 (290 mg, 0.65 mmol),
D-valinol (337 mg, 3.25 mmol), and DIEA (560 .mu.L, 3.25 mmol) in
EtOH (5 mL). Purification via flash chromatography (2%
MeOH/CH.sub.2Cl.sub.2) afforded the product as a clear oil (200 mg,
0.49 mmol, 75% yield); R.sub.f=0.26 (2% MeOH/CH.sub.2Cl.sub.2);
LRMS (m/z) [M+1].sup.+ 412.5 (calculated for
C.sub.22H.sub.38NO.sub.6, 412.5).
Example 8
2-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylox-
ycarbonylamino}-3,3-dimethyl-butyric acid methyl ester
##STR00041##
General procedure A was followed using 1 (65 mg, 0.15 mmol), D-tBu
glycine methyl ester hydrochloride (132 mg, 0.73 mmol), and DIEA
(127 .mu.L, 0.73 mmol) in EtOH (8 mL). Purification via flash
chromatography (10% EtOAc/CH.sub.2Cl.sub.2) afforded the product as
a clear oil (10 mg, 0.02 mmol, 15% yield); R.sub.f=0.22 (10%
EtOAc/CH.sub.2Cl.sub.2); LRMS (m/z) [M+1].sup.+ 454.5 (calculated
for C.sub.24H.sub.40NO.sub.7, 454.5).
Example 9
Cyclohexyl-2-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylox-
ycarbonylamino}-acetic acid methyl ester
##STR00042##
General procedure A was followed using 1 (65 mg, 0.15 mmol),
D-cyclohexyl glycine methyl ester hydrochloride (207 mg, 0.73
mmol), and DIEA (127 .mu.L, 0.73 mmol) in EtOH (7 mL). Purification
via flash chromatography (10% EtOAc/CH.sub.2Cl.sub.2) afforded the
product as a clear oil (20 mg, 0.04 mmol, 28% yield); R.sub.f=0.22
(10% EtOAc/CH.sub.2Cl.sub.2); LRMS (m/z) [M+1].sup.+ 480.3
(calculated for C.sub.26H.sub.42NO.sub.7, 480.3).
Example 10
2-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yloxy-
carbonylamino}-3-methyl-pentanoic acid methyl ester
##STR00043##
General procedure A was followed using 1 (65 mg, 0.15 mmol),
D-isoleucine methyl ester hydrochloride (132 mg, 0.73 mmol), and
DIEA (127 .mu.L, 0.73 mmol) in EtOH (7 mL). Purification via flash
chromatography (10% EtOAc/CH.sub.2Cl.sub.2) afforded the product as
a clear oil (20 mg, 0.04 mmol, 30% yield); R.sub.f=0.20 (10%
EtOAc/CH.sub.2Cl.sub.2); LRMS (m/z) [M+1].sup.+ 454.5 (calculated
for C.sub.24H.sub.40NO.sub.7, 454.5).
Example 11
[1-(1-Carbamoyl-2-hydroxy-ethylcarbamoyl)-2-methyl-propyl]-carbamic
acid-(3R, 4S, 5S, 6R)-5-methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl]-oxiranyl-1-oxa-spiro[2.5]oct-6-yl
ester
##STR00044##
General procedure A was followed using 1 (74 mg, 0.17 mmol),
H-D-vS-NH.sub.2.TFA (262 mg, 0.83 mmol), and DIEA (140 .mu.L, 0.83
mmol) in EtOH (5 mL). Purification via HPLC (60%
CH.sub.3CN/H.sub.2O) afforded the as a white solid (34 mg, 0.07
mmol, 40% yield); R.sub.f=0.21 (5% MeOH/CH.sub.2Cl.sub.2); LRMS
(m/z) [M+1].sup.+ 512.5 (calculated for
C.sub.25H.sub.42N.sub.3O.sub.8, 512.3).
Example 12
2-(3-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yl
}-ureido)-3-methyl-butyramide
##STR00045##
(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylam-
ine (3; PCT Publication No. WO 99/59987) was prepared according to
the published procedure. To a solution of crude 3 (29 mg, 0.1
mmol), DIEA (21 mL, 0.1 mmol) and DMAP (2 mg) in CH.sub.2Cl.sub.2
(1.5 mL) cooled to 0.degree. C. was added p-NO.sub.2 phenyl
chloroformate (25 mg, 0.12 mmol). After 45 minutes, the reaction
was warmed to room temperature and a solution of
H-D-val-NH.sub.2.HCl (40 mg, 0.15 mmol) in EtOH (1 mL) and DIEA (35
.mu.L, 0.2 mmol) was added.
The reaction was continued for 1 hour, then was concentrated in
vacuo, taken up into EtOAc (15 mL), and washed with dilute
HCl.sub.aq (2.times.15 mL), H.sub.2O (2.times.15 mL) and brine (15
mL). Purification via flash chromatography (5%
MeOH/CH.sub.2Cl.sub.2) afforded the product as a white solid (6 mg,
0.014 mmol, 13% yield from 3); R.sub.f=0.12 (5%
MeOH/CH.sub.2Cl.sub.2); LRMS (m/z) [M+1].sup.+ 424.4 (calculated
for C.sub.22H.sub.38N.sub.3O.sub.5, 424.4).
Example 13
N-Carbamoyl (ID#31) 3R, 4S, 5S, 6R)
5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-butyl)-oxiranyl]-1-oxa-spiro[2.5-
]oct-6-yl ester
##STR00046##
General Procedure B, Part I was followed using 2 (41 mg, 0.11
mmol), PS-DCC (256 mg, 0.28 mmol) in DMF (1 mL) and
H-RGD(Bn)S(OBn)P--NH.sub.2.2TFA (37 mg, 0.04 mmol), NMM (4 .mu.L,
0.04 mmol) in DMF (0.5 mL). Purification via HPLC (70%
CH.sub.3CN/H.sub.2O/0.075% TFA) afforded the product as a white
floculent solid (9.3 mg, 0.009 mmol, 17% yield); LRMS (m/z)
[M+1].sup.+ 1075.4 (calculated for
C.sub.53H.sub.75N.sub.10O.sub.14, 1075.5).
General Procedure, Part II was followed using the product in Part I
(9.3 mg, 0.009 mmol) and Pd/C (2 mg) in MeOH (1 mL), and a H.sub.2
atmosphere (38 psi) for 24 hours. Purification via HPLC (55%
CH.sub.3CN/H.sub.2O/0.075% TFA) afforded the product as a white
solid (5 mg, 0.006 mmol, 65% yield); LRMS (m/z) [M+1].sup.+ 897.3
(calculated for C.sub.39H.sub.65N.sub.10O.sub.14, 897.5).
Example 14
N-Carbamoyl (ID#30) 3R, 4S, 5S, 6R)
5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-butyl)-oxiranyl]-1-oxa-spiro[2.5-
]oct-6-yl ester
##STR00047##
General Procedure, Part I was followed using 2 (38 mg, 0.10 mmol)
and PS-DCC (238 mg, 0.25 mmol) in DMF (1 mL),
H-RGD(Bn)Y(OMe)RE(Bn)-NH.sub.2.3TFA (35 mg, 0.03 mmol) and NMM (3
.mu.L, 0.03 mmol) in DMF (0.5 mL). Purification via HPLC (70%
CH.sub.3CN/H.sub.2O/0.075% TFA) afforded the product as a white
floculent solid (4.0 mg, 0.002 mmol, 8% yield); LRMS (m/z)
[M+2/2].sup.+ 677.6 (calculated for C.sub.66H.sub.92N14O.sub.17,
677.8).
General Procedure, Part II was followed using the product in Part I
(3.0 mg, 0.002 mmol) and Pd/C (2 mg) in MeOH (1 mL), under a
H.sub.2 atmosphere (38 psi) for 24 hours. Purification via HPLC
(55% CH.sub.3CN/H.sub.2O/0.075% TFA) afforded the product as a
white solid (3.3 mg, 0.0027 mmol, 94% yield); LRMS (m/z)
[M+2/2].sup.+ 588.5 (calculated for C.sub.52H.sub.82N14O.sub.17,
588.7).
Example 15
N-Carbamoyl (ID#32) 3R, 4S, 5S, 6R)
5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-butyl)-oxiranyl]-1-oxa-spiro[2.5-
]oct-6-yl ester
##STR00048##
General Procedure B, Part I was followed using 2 (38 mg, 0.10
mmol), PS-DCC (238 mg, 0.25 mmol), and HOBt (29 mg, 0.25 mmol) in
DMF (1 mL), and H-RGD(Bn)NH.sub.2.TFA (29 mg, 0.04 mmol) and NMM
(4.8 .mu.L, 0.04 mmol) in DMF (0.5 mL). Purification via HPLC (60%
CH.sub.3CN/H.sub.2O/0.075% TFA) afforded the product as a white
solid (35 mg, 0.04 mmol, 44% yield); LRMS (m/z) 801.2 (calculated
for C.sub.38H.sub.57N.sub.8O.sub.11, 801.4).
General Procedure, Part II was followed using the product in Part I
(35 mg, 0.04 mmol) and Pd/C (2 mg) in MeOH (1 mL), under a H.sub.2
atmosphere (38 psi) for 24 hours. Purification via HPLC (50%
CH.sub.3CN/H.sub.2O/0.075% TFA) afforded the product as a white
solid (22 mg, 0.03 mmol, 71% yield); LRMS (m/z) 713.2 (calculated
for C.sub.31H.sub.53N.sub.8O.sub.11, 713.4).
Example 16
N-Carbamoyl (ID#40) (3R, 4S, 5S, 6R)
5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spir-
o[2.5]oct-6-yl ester
##STR00049##
General Procedure B, Part I was followed using 2 (65 mg, 0.17
mmol), PS-DCC (405 mg, 0.43 mmol), and HOBt (34 mg, 0.26 mmol) in
DMF (1 mL), and H-RG(pyridyl)D-OMe (43 mg, 0.06 mmol) and NMM (7
.mu.L, 0.06 mmol) in DMF (0.5 mL). Purification via HPLC (50%
CH.sub.3CN/H.sub.2O/0.075% TFA) afforded the product as a white
solid (15 mg, 0.02 mmol, 34% yield); LRMS (m/z) 773.2 (calculated
for C.sub.34H.sub.55N.sub.4O.sub.10, 773.4).
The product of Part I (11 mg, 0.01 mmol) was dissolved into
THF:MeOH:H.sub.2O (2:1:1, 500 .mu.L) and treated with LiOH.H.sub.2O
(1.2 mg, 0.02 mmol) for 2 hours. The crude material was diluted
with EtOAc (5 mL) and acidified with dilute HCl (10 mL). The
aqueous phase was washed with additional EtOAc (2.times.5 mL), the
combined organic extracts dried over Na.sub.2SO.sub.4 and the
solvent removed in vacuo. Purification via HPLC (30%
CH.sub.3CN/H.sub.2O/0.075% TFA) afforded the product as a white
solid (2 mg, 0.003 mmol, 19% yield). LRMS (m/z) 745.3 (calculated
for C.sub.33H.sub.53N.sub.4O.sub.10, 745.4).
Example 17
N-Carbamoyl (ID#39) (3R, 4S, 5S, 6R)
5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spir-
o[2.5]oct-6-yl ester
##STR00050##
General procedure B, Part I was followed using 2 (25 mg, 0.07 mmol)
and PS-DCC (155 mg, 0.16 mmol) in DMF (1 mL), and
H-PLGMWAG-NH.sub.2 (20 mg, 0.03 mmol) and NMM (3 .mu.L, 0.03 mmol)
in DMF (0.5 mL). Purification via HPLC (70%
CH.sub.3CN/H.sub.2O/0.075% TFA) afforded the product as a white
solid (1.4 mg, 0.001 mmol, 5% yield); LRMS (m/z) [M+1]+ 1095.6
(calculated for C.sub.53H.sub.79N.sub.10O.sub.13S, 1095.6).
Example 18
N-Carbamoyl (ID#26) (3R, 4S, 5S, 6R)
5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spir-
o[2.5]oct-6-yl ester
##STR00051##
General Procedure B, Part I was followed using 2 (69 mg, 0.18
mmol), PS-DCC (429 mg, 0.45 mmol) and HOBt (21 mg, 0.18 mmol) in
DMF (1 mL), and H-PL(N-Me)G-OMe (31 mg, 0.07 mmol) and NMM (8
.mu.L, 0.07 mmol) in DMF (0.5 mL). Purification via HPLC (70%
CH.sub.3CN/H.sub.2O/0.075% TFA) afforded the product as a white
solid (27 mg, 0.04 mmol, 59% yield); LRMS (m/z) [M+1]+ 679.4
(calculated for C.sub.34H.sub.55N.sub.4O.sub.10, 679.4).
The product of Part I (27 mg, 0.04 mmol) was dissolved into
THF:MeOH:H.sub.2O (2:1:1, 1.5 mL) and treated with LiOH.H.sub.2O (4
mg, 0.10 mmol) for 1 hour. The solution was acidified to pH 3 using
0.1 N HCl, and the MeOH and THF removed in vacuo. Purification via
HPLC (60% CH.sub.3CN/H.sub.2O/0.075% TFA) afforded the product as a
white solid (6 mg, 0.01 mmol, 23% yield). LRMS (m/z) 665.4
(calculated for C.sub.33H.sub.53N.sub.4O.sub.10, 665.4).
Example 19
N-Carbamoyl (ID#27) (3R, 4S, 5S, 6R)
5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spir-
o[2.5]oct-6-yl ester
##STR00052##
General Procedure B, Part I was followed using 2 (44 mg, 0.12
mmol), PS-DCC (276 mg, 0.29 mmol) and HOBt (27 mg, 0.23 mmol) in
DMF (1 mL), and H-PLG-OMe (20 mg, 0.05 mmol) and NMM (5 .mu.L, 0.05
mmol) in DMF (0.5 mL). Purification via HPLC (90%
CH.sub.3CN/H.sub.2O/0.075% TFA) afforded the product as a white
solid (13 mg, 0.02 mmol, 43% yield); LRMS (m/z) [M+1]+ 664.4
(calculated for C.sub.34H.sub.52N.sub.4O.sub.10, 664.4).
The product in Part I (27 mg, 0.04 mmol) was dissolved into
THF:MeOH:H.sub.2O (2:1:1, 790 .mu.L) and treated with LiOH.H.sub.2O
(1.2 mg, 0.03 mmol) for 2 hours. The solution was acidified to pH 3
using 0.1 N HCl, and the MeOH and THF removed in vacuo.
Purification via HPLC (90% CH.sub.3CN/H.sub.2O/0.075% TFA) afforded
the product as a white solid (1.8 mg, 0.003 mmol, 15% yield). LRMS
(m/z) 650.4 (calculated for C.sub.32H.sub.50N.sub.4O.sub.10,
650.4).
Example 20
(ID#24)-(2R-{(3R, 4S, 5S, 6R)
5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spir-
o[2.5]oct-6-yloxycarbonyl}amino-3-methyl-butanol)ester
##STR00053##
General Procedure C was followed using the compound in Example 7
(189 mg, 0.46 mmol), Ac-PLGMWA-OH (329 mg, 0.46 mmol), DMAP (84 mg,
0.69 mmol) and DIC (72 .mu.L, 0.46 mmol) in CH.sub.2Cl.sub.2 (5
mL). After 18 hours, the solvent was removed in vacuo and
purification via flash chromatography (2% MeOH/CH.sub.2Cl.sub.2)
afforded the product as a white solid (357 mg, 0.32 mmol, 70%
yield); R.sub.f=0.18 (5% MeOH/CH.sub.2Cl.sub.2); LRMS (m/z)
[M+1].sup.+ 1110.3 (calculated for
C.sub.56H.sub.85N.sub.8O.sub.13S, 1110.3).
Example 21
(ID#36)-(2R-{(3R, 4S, 5S, 6R)
5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spir-
o[2.5]oct-6-yloxycarbonyl}amino-3-methyl-butanol)ester
##STR00054##
General Procedure C was followed using the compound in Example 7
(61 mg, 0.15 mmol), Ac-PLGMG-OH (92 mg, 0.18 mmol), DMAP (22 mg,
0.18 mmol) and DIC (28 .mu.L, 0.18 mmol) in CH.sub.2Cl.sub.2 (2
mL). After 7 hours, the solvent was removed in vacuo and
purification via flash chromatography (3% MeOH/CH.sub.2Cl.sub.2)
afforded the product as a white solid (61 mg, 0.0 mmol, 45% yield);
R.sub.f=0.20 (5% MeOH/CH.sub.2Cl.sub.2); LRMS (m/z) [M+1].sup.+
909.7 (calculated for C.sub.44H.sub.73N.sub.6O.sub.12S, 909.5).
Example 22
(ID#37)-(2R-{(3R, 4S, 5S, 6R)
5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spir-
o[2.5]oct-6-yloxycarbonyl}amino-3-methyl-butanol)ester
##STR00055##
General Procedure C was followed using the compound in Example 7
(79 mg, 0.19 mmol), Fmoc-MWA-OH (121 mg, 0.19 mmol) and DMAP (4 mg,
0.03 mmol) and DIC (30 .mu.L, 0.19 mmol) in CH.sub.2Cl.sub.2 (2
mL). After 11 hours, the solvent was removed in vacuo and
purification via flash chromatography (2% MeOH/CH.sub.2Cl.sub.2)
afforded the product as a white solid (128 mg, 0.12 mmol, 65%
yield); LRMS (m/z) [M+1].sup.+ 1022.9 (calculated for
C.sub.44H.sub.73N.sub.6O.sub.12S, 1022.5).
The product from General Procedure C (above) (54 mg, 0.05 mmol) was
dissolved into anhydrous CH.sub.2Cl.sub.2 (3 mL) cooled to
0.degree. C., then treated with a gentle stream of NH.sub.3(g) for
15 minutes. The reaction was sealed and continued at 0.degree. C.
for 36 hours. The solvent was removed in vacuo, and the crude
residue acidified with CH.sub.3CN/H.sub.2O (0.075% TFA) (5 mL).
Purification via HPLC (70% CH.sub.3CN/H.sub.2O/0.075% TFA) afforded
the product as a white solid (2 mg, 0.003 mmol, 5% yield); LRMS
(m/z) [M+1].sup.+ 800.6 (calculated for
C.sub.41H.sub.62N.sub.5O.sub.9S, 800.5).
Example 23
(ID#38)-(2R-{(3R, 4S, 5S, 6R)
5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spir-
o[2.5]oct-6-yloxycarbonyl}amino-3-methyl-butanol)ester
##STR00056##
General Procedure C was followed using the compound in Example 7
(76 mg, 0.18 mmol), Fmoc-MG-OH (79 mg, 0.18 mmol) and DMAP (4 mg,
0.03 mmol) and VDIC (29 .mu.L, 0.18 mmol) in CH.sub.2Cl.sub.2 (2
mL). After 10 hours, the solvent was removed in vacuo and
purification via flash chromatography (2% MeOH/CH.sub.2Cl.sub.2)
afforded the product as a white solid (128 mg, 0.12 mmol, 65%
yield); LRMS (m/z) [M+1].sup.+ 822.6 (calculated for
C.sub.44H.sub.60N.sub.3O.sub.10S, 822.5).
The product from General Procedure C (above) (42 mg, 0.05 mmol) was
dissolved into anhydrous CH.sub.2Cl.sub.2 (3 mL) cooled to
0.degree. C., then treated with a gentle stream of NH.sub.3(g) for
15 minutes. The reaction was sealed and continued at 0.degree. C.
for 36 hours. The solvent was removed in vacuo, and the crude
residue acidified with CH.sub.3CN/H.sub.2O (0.075% TFA) (5 mL).
Purification via HPLC (70% CH.sub.3CN/H.sub.2O/0.075% TFA) afforded
the product as a white solid (2 mg, 0.003 mmol, 5% yield); LRMS
(m/z) [M+1].sup.+ 600.4 (calculated for
C.sub.29H.sub.50N.sub.3O.sub.8S, 600.4).
Example 24
2-{(3R, 4S, 5S, 6R)-5-Methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylox-
ycarbonylamino}-3-methyl-butyric acid
##STR00057##
The compound in Example 2 (9 mg, 0.02 mmol) was dissolved into
THF:MeOH:H.sub.2O (1 mL) and treated with LiOH.H.sub.2O (2 mg, 0.05
mmol). After 2 hours, the reaction was partitioned between EtOAc (5
mL) and dilute HCl (5 mL). The organic phase was dried over
Na.sub.2SO.sub.4 and the solvent removed in vacuo. Purification via
HPLC (85% CH.sub.3CN/H.sub.2O/0.075% TFA) afforded the product as a
white solid (0.58 mg, 0.001 mmol, 6% yield); LRMS (m/z) [M+1].sup.+
426.4 (calculated for C.sub.22H.sub.36NO.sub.7, 426.5).
Example 25
(ID#34)-(2R-{(3R, 4S, 5S, 6R)
5-methoxy-4-[(2R,3R)2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spir-
o[2.5]oct-6-yloxycarbonyl}amino-3-methyl-butanol)ester
##STR00058##
General Procedure C was followed using the compound in Example 7
(41 mg, 0.10 mmol), Ac-PLGMG-OH (63 mg, 0.12 mmol), DMAP (15 mg,
0.12 mmol) and DIC (19 .mu.L, 0.12 mmol) in CH.sub.2Cl.sub.2 (2
mL). After 7 hours, the solvent was removed in vacuo and
purification via flash chromatography (3% MeOH/CH.sub.2Cl.sub.2)
afforded the product as a white solid (43 mg, 0.05 mmol, 47%
yield); R.sub.f=0.21 (5% MeOH/CH.sub.2Cl.sub.2); LRMS (m/z)
[M+1].sup.+ 923.7 (calculated for C.sub.45H.sub.75N.sub.6O.sub.12S,
923.5).
Example 26
The angiogenesis inhibitor compounds of the invention were tested
for their ability to modulate human endothelial cell growth and for
their ability to modulate the activity of MetAP2. The MetAP2 enzyme
assay was performed essentially as described in Turk, B. et al.
(1999) Chem. & Bio. 6: 823-833, the entire contents of which
are incorporated herein by reference. The bovine aortic endothelial
cell growth assay (Baec assay) was performed essentially as
described in Turk, B. et al. (supra), the entire contents of which
are incorporated herein by reference.
For the human endothelial cell growth assay, human umbilical vein
endothelial cells (HUVEC) were maintained in Clonetics endothelial
growth medium (EGM) in a 37.degree. C. humidified incubator. Cells
were detached with trypsin and pelleted by centrifugation at
300.times.g for 5 minutes at room temperature. HUVEC were added to
96-well plates at 5,000 cells/well. After incubating for 6 hours,
the medium was replaced with 0.2 ml fresh EGM supplemented with 0.5
nM bFGF and the desired concentration of test angiogenesis
inhibitor compound. Test angiogenesis inhibitor compounds were
initially dissolved in ethanol at stock concentrations of either 10
mM or 0.1 mM, and subsequently diluted in EGM to obtain
concentrations from 1 pM to 10 .mu.M. After 48 hours at 37.degree.
C., the medium was replaced with fresh bFGF-supplemented EGM and
test angiogenesis inhibitor compound. Following incubation for an
additional 48 hours at 37.degree. C. MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) was
added to 1 mg/ml. After 2-4 hours at 37.degree. C. the medium was
replaced with 0.1 ml/well isopropanol. The plates were placed on a
shaker for 15 minutes at room temperature and analyzed in a
Labsystems Multiskan plate spectrophotometer at an optical density
of 570 nm.
The results of the assays, set forth below in Tables I-III,
demonstrate that the angiogeness inhibitor compounds of the
invention have excellent MetAP2 inhibitory activity and are able to
inhibit endothelial cell growth at the picomolar range.
TABLE-US-00001 TABLE I MetAP2 Assay Example IC.sub.50 (nM) 1 4.7 2
2 3 5.5 4 2.7 13 2.9 14 4000 17 16.7
TABLE-US-00002 TABLE II Huvec Assay Example IC.sub.50 (pM) 1 18 2
40 3 38 4 36 5 93 13 (>10 .mu.M) 14 (>10 .mu.M) 15 (>10
.mu.M) 17 (95 nM) 18 (>100 nM) 19 (>100 nM) 24 5444
TABLE-US-00003 TABLE III Baec Assay Example IC.sub.50 (pM) 1 17 2
48 3 118 4 35 5 46 6 220 7 128 8 313 9 165 10 179 11 (>100 nM)
16 (>100 nM) 19 (>100 nM) 22 326 23 207
The identity of the angiogenesis inhibitor compounds used in each
of the experiments is shown in Tables IV and V below.
TABLE-US-00004 TABLE IV ##STR00059## Example ID# Sequence 13 31
X-GlyArgGlyAspSerPro-NH2 14 30 X-GlyArgGlyAspTyr(OMe)ArgGlu-NH2 15
32 X-GlyArgGlyAsp-NH2 16 40
X-Gly-Arg-Gly-(3-amino-3-pyridylpropionic acid) 17 39
X-GlyProLeuGlyMetTrpAlaGly-NH2 18 26 X-GlyProLeuSar-OH 19 27
X-GlyProLeuGly-OH
TABLE-US-00005 TABLE V ##STR00060## Example ID# Sequence 20 24
Ac-ProLeuGly-MetTrpAla-Y 21 36 Ac-ProLeuGlyMetGly-Y 22 37
H-MetTrpAla-Y 23 38 H-MetGly-Y 25 34 Ac-ProLeuGlyMetAla-Y
Example 27
The compound of example 5 was also evaluated against a panel of
cancer cell lines (Alley, M. C. et al. (1998) Cancer Research 48:
589-601; Grever, M. R., et al. (1992) Seminars in Oncology, Vol.
19, No. 6, pp 622-638; Boyd, M. R., and Paull, K. D. (1995) Drug
Development Research 34: 91-109). The human tumor cell lines of the
cancer screening panel were grown in RPMI 1640 medium containing 5%
fetal bovine serum and 2 mM L-glutamine. Cells were inoculated into
96 well microtiter plates in 100 .mu.L at plating densities ranging
from 5,000 to 40,000 cells/well depending on the doubling time of
individual cell lines. After cell inoculation, the microtiter
plates were incubated at 37.degree. C., 5% CO.sub.2, 95% air and
100% relative humidity for 24 hours prior to addition of
experimental drugs.
After the 24 hour incubation period, two plates of each cell line
were fixed in situ with TCA, to represent a measurement of the cell
population for each cell line at the time of drug addition (Tz).
Experimental drugs were solubilized in dimethyl sulfoxide at
400-fold the desired final maximum test concentration and stored
frozen prior to use. At the time of drug addition, an aliquot of
frozen concentrate was thawed and diluted to twice the desired
final maximum test concentration with complete medium containing 50
.mu.g/ml gentamicin. Additional four, 10-fold or 1/2 log serial
dilutions were made to provide a total of five drug concentrations
plus control. Aliquots of 100 .mu.l of these different drug
dilutions were added to the appropriate microtiter wells already
containing 100 .mu.l of medium, resulting in the required final
drug concentrations.
Following drug addition, the plates were incubated for an
additional 48 hours at 37.degree. C., 5% CO.sub.2, 95% air, and
100% relative humidity. For adherent cells, the assay was
terminated by the addition of cold TCA. Cells were fixed in situ by
the gentle addition of 50 .mu.l of cold 50% (w/v) TCA (final
concentration, 10% TCA) and incubated for 60 minutes at 4.degree.
C. The supernatant was discarded, and the plates were washed five
times with tap water and air dried. Sulforhodamine B (SRB) solution
(100 .mu.l) at 0.4% (w/v) in 1% acetic acid were added to each
well, and plates were incubated for 10 minutes at room temperature.
After staining, unbound dye was removed by washing five times with
1% acetic acid and the plates were air dried. Bound stain was
subsequently solubilized with 10 mM trizma base, and the absorbance
was read on an automated plate reader at a wavelength of 515 nm.
For suspension of the cells, the methodology used was the same
except that the assay was terminated by fixing settled cells at the
bottom of the wells by gently adding 50 .mu.l of 80% TCA (final
concentration, 16% TCA). Using the seven absorbance measurements
[time zero, (Tz), control growth, (C), and test growth in the
presence of drug at the five concentration levels (Ti)], the
percentage growth was calculated at each of the drug concentrations
levels.
Percentage growth inhibition was calculated as:
[(Ti-Tz)/(C-Tz)].times.100 for concentrations for which Ti>/=Tz
[(Ti-Tz)/Tz].times.100 for concentrations for which Ti<Tz.
Growth inhibition of 50% (GI.sub.50) was calculated from
[(Ti-Tz)/(C-Tz)].times.100=50, which is the drug concentration
resulting in a 50% reduction in the net protein increase (as
measured by SRB staining) in control cells during the drug
incubation. The GI.sub.50 was calculated for each of the cell lines
if the level of activity is reached; however, if the effect was not
reached or is exceeded, the value for that parameter is expressed
as greater or less than the maximum (10.sup.-4 M) or minimum
(10.sup.-8 M) concentration tested.
TABLE-US-00006 TABLE VI Effect of the compound of example 5 on
tumor cell line panel Cell line Tumor type GI.sub.50 (moles
liter.sup.-1) HL-60(TB) Leukemia 2.17 .times. 10.sup.-5 K-562
Leukemia 6.44 .times. 10.sup.-5 MOLT-4 Leukemia 3.56 .times.
10.sup.-5 RPMI-8226 Leukemia <1 .times. 10.sup.-8 SR Leukemia
<1 .times. 10.sup.-8 EKVX Non-Small Cell Lung 2.08 .times.
10.sup.-5 HOP-62 Non-Small Cell Lung <1 .times. 10.sup.-8 HOP-92
Non-Small Cell Lung 3.39 .times. 10.sup.-5 NCI-H226 Non-Small Cell
Lung 7.91 .times. 10.sup.-7 NCI-H23 Non-Small Cell Lung 6.34
.times. 10.sup.-6 NCI-H322M Non-Small Cell Lung 4.68 .times.
10.sup.-8 NCI-H460 Non-Small Cell Lung <1 .times. 10.sup.-8
NCI-H522 Non-Small Cell Lung 1.29 .times. 10.sup.-5 COLO 205 Colon
<1 .times. 10.sup.-8 HCT-116 Colon <1 .times. 10.sup.-8
HCT-15 Colon 7.13 .times. 10.sup.-6 HT29 Colon 1.61 .times.
10.sup.-5 KM12 Colon <1 .times. 10.sup.-8 SW-620 Colon >1
.times. 10.sup.-4 SF-268 CNS 2.61 .times. 10.sup.-5 SF-295 CNS
<1 .times. 10.sup.-8 SF-539 CNS 2.06 .times. 10.sup.-5 SNB-19
CNS <1 .times. 10.sup.-8 SNB-75 CNS 9.09 .times. 10.sup.-5
MALME-3M Melanoma 5.31 .times. 10.sup.-8 M14 Melanoma <1 .times.
10.sup.-8 SK-MEL-2 Melanoma >1 .times. 10.sup.-4 SK-MEL-28
Melanoma 5.96 .times. 10.sup.-6 SK-MEL-5 Melanoma >1 .times.
10.sup.-4 UACC-257 Melanoma 1.48 .times. 10.sup.-6 UACC-62 Melanoma
<1 .times. 10.sup.-8 IGR-OV1 Ovarian <1 .times. 10.sup.-8
OVCAR-3 Ovarian 4.18 .times. 10.sup.-5 OVCAR-4 Ovarian 3.66 .times.
10.sup.-5 OVCAR-5 Ovarian 1.35 .times. 10.sup.-8 OVCAR-8 Ovarian
1.84 .times. 10.sup.-5 SK-OV-3 Ovarian 7.37 .times. 10.sup.-6 786-0
Renal 1.61 .times. 10.sup.-5 A498 Renal >1 .times. 10.sup.-4
ACHN Renal <1 .times. 10.sup.-8 CAKI-1 Renal <1 .times.
10.sup.-8 RXF 393 Renal 4.02 .times. 10.sup.-5 SN12C Renal <1
.times. 10.sup.-8 TK-10 Renal 5.43 .times. 10.sup.-8 PC-3 Prostate
1.80 .times. 10.sup.-5 DU-145 Prostate <1 .times. 10.sup.-8 MCF7
Breast 1.24 .times. 10.sup.-5 NCI/ADR-RES Breast 3.42 .times.
10.sup.-5 MDA-MB-231/ATCC Breast <1 .times. 10.sup.-8 HS 578T
Breast 1.15 .times. 10.sup.-6 MDA-N Breast 1.58 .times.
10.sup.-6
Results
The results of the cell line screen, presented in Table VI, show
that the compound of example 5 has a significant inhibitory effect
on a wide variety of tumor cell lines. The results also show that
certain cell lines are much more sensitive to the compound of
example 5 than are others, indicating that this compound is
selective for certain cell lines.
EXAMPLES
In Examples 28-30, the compound of Example 5 (hereinafter "Compound
5") was used:
(1-Carbamoyl-2-methyl-propyl)-carbamic acid-(3R, 4S, 5S,
6R)-5-methoxy-4-[(2R,
3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yl
ester
##STR00061##
Example 28
Inhibition of B-Cell Lymphoma Cell Line in Culture
Objective:
To determine the inhibition of germinal center derived B cell
lymphoma lines by Compound 5.
Experimental Design:
Compound 5 was incubated at final concentrations ranging from
0.01-100 nM with 50,000 cells/mL of germinal center derived B cell
lymphoma lines. Incubations lasted for five or six days after which
cell numbers were determined from triplicate flasks at each
concentration.
Results:
Compound A inhibited the proliferation of all lymphoma lines tested
except for the Ramos line, a Burkitt's lymphoma cell line. Table
VII shows the maximum growth inhibition and estimated concentration
producing a 50% decrease in cell proliferation (GI.sub.50%) in
Compound A treated cultures relative to growth observed in vehicle
control cultures.
TABLE-US-00007 TABLE VII Inhibition of GC Derived B Cell Lymphoma
Lines Growth Inhibition by Compound B Lymphoma A Relative to
Vehicle Control Cell Line Classification.sup.1 Maximum Inhibition
GI.sub.50% SU-DHL-16 DLBCL .sup. 60%.sup.2 1.9 nM Pfeiffer DLBCL
54% 0.27 nM DB DLBCL .sup. 42%.sup.2 -- D10 FL 59% 0.42 nM H2 FL
59% 0.16 nM Ramos BL -- -- ST486 BL 53% 0.22 nM
.sup.1DLBCL--diffuse large B cell lymphoma, FL--follicular
lymphoma, BL--Burkitt's lymphoma. .sup.2Cell number determined at
day 5 due to rapid growth of vehicle treated cultures
Conclusion:
Compound 5 inhibited the proliferation of all DLBCL and FL cell
lines tested at low nanomolar concentrations.
Example 29
Inhibition of SR Cell Line in Culture
Objective:
To evaluate the dose response inhibition of the human SR
lymphoblast cell line in culture
Experimental Design:
Compound 5 was incubated at final concentrations ranging from 0.1
nM to 10 .mu.M with 25,000 cells/mL of human lymphoblast SR cells.
Incubations were conducted for 3, 5 or 6 days after which cell
proliferation relative to vehicle treatment was determined using a
.sup.3H-thymidine incorporation assay. Medium was replaced and
fresh drug was added on day 3 for the 5 and 6 day assays.
Results:
FIG. 1 shows representative data from these cell proliferation
assays. Compound 5 inhibited proliferation of the SR cell line by
59-75% at concentrations from 1-100 nM with a mean GI.sub.50 of 0.5
nM in the 5 and 6 day assays.
Conclusion:
These results demonstrate that Compound 5 can inhibit proliferation
of the SR cell line in culture at nM concentrations. Maximal
inhibition by Compound 5 was greater than 90% with a mean
GI.sub.50% of 0.5 nM following five or six days of exposure to
Compound 5.
Example 30
Evaluation of In Vivo Efficacy of Compound 5 in SR Lymphoma Cell
Tumors Grown in Mice
Objective:
This study was performed to determine the in vivo efficacy for
Compound A administered either subcutaneously or orally in SR
tumor-bearing mice
Experimental Design:
SR lymphoma tumor cells were injected subcutaneously into SCID/NCr
female mice. Tumors were measured using a caliper every 3-4 days
beginning on day 12 post-implantation of tumor cells. Animals were
weighed routinely on the same days as tumor measurments and
monitored for clinical signs of any adverse, drug-related side
effects. Treatment with Compound 5 or vehicle began on Day 12 for 5
weeks and ended on Day 44. The endpoints evaluated were optimal
percent treated/control (% T/C) and tumor growth delay measured at
multiple timepoints during the study.
Results:
In SR tumor xenografts both oral and subcutaneous routes of
administration of Compound 5 significantly suppressed tumor growth
in a dose-dependent manner and the oral route appeared to be
slightly superior to the subcutaneous route in this model (FIG. 2).
Compound 5 administered subcutaneously at 15 or 30 mg/kg produced
optimal % T/C values of 70 or 50, respectively, whereas
administration by the oral route achieved optimal % T/C values of
48 or 43 at the 15 or 30 mg/kg dose, respectively. Moreover, oral
administration of Compound 5 was more efficacious than subcutaneous
administration, as determined by tumor growth delay. Compound 5
produced tumor growth delays of 29 or 28% when administered orally
(15 or 30 mg/kg, respectively) compared to 18 or 19% when
administered by the subcutaneous route (15 or 30 mg/kg,
respectively).
Conclusion:
Compound 5 produces a dose-dependent inhibition of growth of SR
lymphoma tumors growing in mice with maximal efficacy observed in
this study at 30 mg/kg administered by the oral route.
Equivalents
Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. Such
equivalents are intended to be encompassed by the following
claims.
SEQUENCE LISTINGS
1
37 1 4 PRT Artificial Sequence VARIANT 4 Xaa at position 4 may be
any amino acid 1 Pro Leu Gly Xaa 1 2 5 PRT Artificial Sequence
VARIANT 2 Xaa at position 2 represents L-cyclohexylalanine 2 Pro
Xaa Gly Xaa His 1 5 3 8 PRT Artificial Sequence Description of
Artificial Sequence Motifs 3 Pro Gln Gly Ile Ala Gly Gln Xaa 1 5 4
7 PRT Artificial Sequence Description of Artificial Sequence Motifs
4 Pro Gln Gly Ile Ala Gly Trp 1 5 5 7 PRT Artificial Sequence
Description of Artificial Sequence Motifs 5 Pro Leu Gly Xaa His Ala
Xaa 1 5 6 7 PRT Artificial Sequence Description of Artificial
Sequence Motifs 6 Pro Leu Gly Leu Trp Ala Xaa 1 5 7 7 PRT
Artificial Sequence Description of Artificial Sequence Motifs 7 Pro
Leu Ala Leu Trp Ala Arg 1 5 8 7 PRT Artificial Sequence Description
of Artificial Sequence Motifs 8 Pro Leu Ala Leu Trp Ala Arg 1 5 9 7
PRT Artificial Sequence Description of Artificial Sequence Motifs 9
Pro Leu Ala Tyr Trp Ala Arg 1 5 10 7 PRT Artificial Sequence
Description of Artificial Sequence Motifs 10 Pro Tyr Ala Tyr Trp
Met Arg 1 5 11 6 PRT Artificial Sequence Description of Artificial
Sequence Motifs 11 Pro Xaa Gly Xaa His Ala 1 5 12 4 PRT Artificial
Sequence Description of Artificial Sequence Motifs 12 Pro Leu Ala
Xaa 1 13 4 PRT Artificial Sequence Description of Artificial
Sequence Motifs 13 Pro Leu Gly Leu 1 14 4 PRT Artificial Sequence
Description of Artificial Sequence Motifs 14 Pro Leu Gly Ala 1 15 8
PRT Artificial Sequence Description of Artificial Sequence Motifs
15 Arg Pro Leu Ala Leu Trp Arg Ser 1 5 16 7 PRT Artificial Sequence
Description of Artificial Sequence Motifs 16 Pro Xaa Ala Xaa Xaa
His Ala 1 5 17 7 PRT Artificial Sequence Description of Artificial
Sequence Motifs 17 Pro Xaa Ala Gly Xaa His Ala 1 5 18 9 PRT
Artificial Sequence Description of Artificial Sequence Motifs 18
Pro Lys Pro Gln Gln Phe Phe Gly Leu 1 5 19 6 PRT Artificial
Sequence Description of Artificial Sequence Motifs 19 Pro Lys Pro
Leu Ala Leu 1 5 20 9 PRT Artificial Sequence Description of
Artificial Sequence Motifs 20 Arg Pro Lys Pro Tyr Ala Xaa Trp Met 1
5 21 9 PRT Artificial Sequence Description of Artificial Sequence
Motifs 21 Arg Pro Lys Pro Val Glu Xaa Trp Arg 1 5 22 9 PRT
Artificial Sequence Description of Artificial Sequence Motifs 22
Arg Pro Lys Pro Val Glu Xaa Trp Arg 1 5 23 8 PRT Artificial
Sequence Description of Artificial Sequence Motifs 23 Arg Pro Lys
Pro Leu Ala Xaa Trp 1 5 24 6 PRT Artificial Sequence Description of
Artificial Sequence Motifs 24 Xaa Leu Gly Met Trp Ala 1 5 25 8 PRT
Artificial Sequence Description of Artificial Sequence Motifs 25
Gly Pro Leu Gly Met His Ala Gly 1 5 26 4 PRT Artificial Sequence
Description of Artificial Sequence Motifs 26 Gly Pro Leu Xaa 1 27 4
PRT Artificial Sequence Description of Artificial Sequence Motifs
27 Gly Pro Leu Gly 1 28 5 PRT Artificial Sequence Description of
Artificial Sequence Motifs 28 Gly Met Gly Leu Pro 1 5 29 5 PRT
Artificial Sequence Description of Artificial Sequence Motifs 29
Ala Met Gly Ile Pro 1 5 30 6 PRT Artificial Sequence Description of
Artificial Sequence Motifs 30 Arg Gly Asp Xaa Arg Glu 1 5 31 6 PRT
Artificial Sequence Description of Artificial Sequence Motifs 31
Gly Arg Gly Asp Ser Pro 1 5 32 4 PRT Artificial Sequence
Description of Artificial Sequence Motifs 32 Gly Arg Gly Asp 1 33 5
PRT Artificial Sequence Description of Artificial Sequence Motifs
33 Xaa Leu Gly Met Ala 1 5 34 10 PRT Artificial Sequence
Description of Artificial Sequence Motifs 34 Xaa Gly Asp Ser Pro
Leu Gly Met Trp Ala 1 5 10 35 7 PRT Artificial Sequence Description
of Artificial Sequence Motifs 35 Pro Leu Gly Met Trp Ser Arg 1 5 36
5 PRT Artificial Sequence Description of Artificial Sequence Motifs
36 Pro Leu Gly Met Gly 1 5 37 8 PRT Artificial Sequence Description
of Artificial Sequence Motifs 37 Gly Pro Leu Gly Met Trp Ala Gly 1
5
* * * * *